Methods of treating cancer by administering a soluble receptor comprising a human Fc domain and the Fri domain from human frizzled receptor

ABSTRACT

The present invention relates to compositions and methods for characterizing, diagnosing, and treating cancer. In particular the invention provides the means and methods for the diagnosis, characterization, prognosis and treatment of cancer and specifically targeting cancer stem cells. The present invention provides a soluble FZD receptor comprising an extracellular domain of a human FZD receptor that inhibits growth of tumor cells. The present invention still further provides a soluble receptor comprising a Fri domain of a human FZD receptor that binds a ligand of a human FZD receptor and said soluble receptor is capable of inhibiting tumor growth. The present invention still further provides a method of treating cancer comprising administering a soluble FZD receptor comprising for example, either an extracellular domain of a human FZD receptor or a Fri domain of a human FZD receptor, in an amount effective to inhibit tumor growth.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/266,998, filed May 1, 2014, now U.S. Pat. No. 9,228,013, which is acontinuation of U.S. application Ser. No. 13/667,191, filed Nov. 2,2012, now U.S. Pat. No. 8,765,913, which is a continuation of U.S.application Ser. No. 12/724,169, filed Mar. 15, 2010, now U.S. Pat. No.8,324,361, which is a division of Ser. No. 11/589,931, filed Oct. 31,2006, now U.S. Pat. No. 7,723,477, which claims the benefit of U.S.Prov. Appl. No. 60/731,468 filed Oct. 31, 2005 and U.S. Prov. Appl. No.60/812,966, filed Jun. 13, 2006, each of which is herein incorporated byreference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:2293_0160008_SL.txt; Size: 17,595 bytes; and Date of Creation: Nov. 25,2015) filed on Nov. 30, 2015, is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the field of oncology and providesnovel compositions and methods for diagnosing and treating cancer. Inparticular, the present invention provides antagonists against cancerand in particular against cancer stem cell markers including receptorfusion proteins useful for the study, diagnosis, and treatment of solidtumors.

Background Art

Cancer is one of the leading causes of death in the developed world,resulting in over 500,000 deaths per year in the United States alone.Over one million people are diagnosed with cancer in the U.S. each year,and overall it is estimated that more than 1 in 3 people will developsome form of cancer during their lifetime. Though there are more than200 different types of cancer, four of them—breast, lung, colorectal,and prostate—account for over half of all new cases (Jemal et al.,Cancer J. Clin. 53:5-26 (2003)).

Breast cancer is the most common cancer in women, with an estimate 12%of women at risk of developing the disease during their lifetime.Although mortality rates have decreased due to earlier detection andimproved treatments, breast cancer remains a leading cause of death inmiddle-aged women. Furthermore, metastatic breast cancer is still anincurable disease. On presentation, most patients with metastatic breastcancer have only one or two organ systems affected, but as the diseaseprogresses, multiple sites usually become involved. The most commonsites of metastatic involvement are locoregional recurrences in the skinand soft tissues of the chest wall, as well as in axilla andsupraclavicular areas. The most common site for distant metastasis isthe bone (30-40% of distant metastasis), followed by the lungs andliver. And although only approximately 1-5% of women with newlydiagnosed breast cancer have distant metastasis at the time ofdiagnosis, approximately 50% of patients with local disease eventuallyrelapse with metastasis within five years. At present the mediansurvival from the manifestation of distant metastases is about threeyears.

Current methods of diagnosing and staging breast cancer include thetumor-node-metastasis (TNM) system that relies on tumor size, tumorpresence in lymph nodes, and the presence of distant metastases asdescribed in the American Joint Committee on Cancer, AJCC Cancer StagingManual, Philadelphia, Pa., Lippincott-Raven Publishers, 5th ed. (1997),pp 171-180, and in Harris, J R: “Staging of breast carcinoma” in Harris,J. R., et al., eds., Breast Diseases, Philadelphia, Lippincott (1991).These parameters are used to provide a prognosis and select anappropriate therapy. The morphologic appearance of the tumor can also beassessed but because tumors with similar histopathologic appearance canexhibit significant clinical variability, this approach has seriouslimitations. Finally assays for cell surface markers can be used todivide certain tumors types into subclasses. For example, one factorconsidered in the prognosis and treatment of breast cancer is thepresence of the estrogen receptor (ER) as ER-positive breast cancerstypically respond more readily to hormonal therapies such as tamoxifenor aromatase inhibitors than ER-negative tumors. Yet these analyses,though useful, are only partially predictive of the clinical behavior ofbreast tumors, and there is much phenotypic diversity present in breastcancers that current diagnostic tools fail to detect and currenttherapies fail to treat.

Prostate cancer is the most common cancer in men in the developed world,representing an estimated 33% of all new cancer cases in the U.S., andis the second most frequent cause of death (Jemal et al., CA Cancer J.Clin. 53:5-26 (2003)). Since the introduction of the prostate specificantigen (PSA) blood test, early detection of prostate cancer hasdramatically improved survival rates, and the five year survival ratefor patients with local and regional stage prostate cancers at the timeof diagnosis is nearing 100%. Yet more than 50% of patients willeventually develop locally advanced or metastatic disease(Muthuramalingam et al., Clin. Oncol. 16:505-516 (2004)).

Currently radical prostatectomy and radiation therapy provide curativetreatment for the majority of localized prostate tumors. However,therapeutic options are very limited for advanced cases. For metastaticdisease, androgen ablation with luteinising hormone-releasing hormone(LHRH) agonist alone or in combination with anti-androgens is thestandard treatment. Yet despite maximal androgen blockage, the diseasenearly always progresses with the majority developingandrogen-independent disease. At present there is no uniformly acceptedtreatment for hormone refractory prostate cancer, and chemotherapeuticregimes are commonly used (Muthuramalingam et al., Clin. Oncol.16:505-516 (2004); Trojan et al., Anticancer Res. 25:551-561 (2005)).

Colorectal cancer is the third most common cancer and the fourth mostfrequent cause of cancer deaths worldwide (Weitz et al., 2005, Lancet365:153-65). Approximately 5-10% of all colorectal cancers arehereditary with one of the main forms being familial adenomatouspolyposis (PAP), an autosomal dominant disease in which about 80% ofaffected individuals contain a germline mutation in the adenomatouspolyposis coli (APC) gene. Colorectal carcinoma has a tendency to invadelocally by circumferential growth and elsewhere by lymphatic,hematogenous, transperitoneal, and perineural spread. The most commonsite of extralymphatic involvement is the liver, with the lungs the mostfrequently affected extra-abdominal organ. Other sites of hematogenousspread include the bones, kidneys, adrenal glands, and brain.

The current staging system for colorectal cancer is based on the degreeof tumor penetration through the bowel wall and the presence or absenceof nodal involvement. This staging system is defined by three majorDuke's classifications: Duke's A disease is confined to submucosa layersof colon or rectum; Duke's B disease has tumors that invade through themuscularis propria and may penetrate the wall of the colon or rectum;and Duke's C disease includes any degree of bowel wall invasion withregional lymph node metastasis. While surgical resection is highlyeffective for early stage colorectal cancers, providing cure rates of95% in Duke's A patients, the rate is reduced to 75% in Duke's Bpatients and the presence of positive lymph node in Duke's C diseasepredicts a 60% likelihood of recurrence within five years. Treatment ofDuke's C patients with a post surgical course of chemotherapy reducesthe recurrence rate to 40%-50%, and is now the standard of care forthese patients.

Lung cancer is the most common cancer worldwide, the third most commonlydiagnosed cancer in the United States, and by far the most frequentcause of cancer deaths (Spiro et al., Am. J. Respir. Crit. Care Med.166:1166-1196 (2002); Jemal et al., CA Cancer J. Clin. 53:5-26 (2003)).Cigarette smoking is believed responsible for an estimated 87% of alllung cancers making it the most deadly preventable disease. Lung canceris divided into two major types that account for over 90% of all lungcancers: small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC). SCLC accounts for 15-20% of cases and is characterized by itsorigin in large central airways and histological composition of sheetsof small cells with little cytoplasm. SCLC is more aggressive thanNSCLC, growing rapidly and metastasizing early and often. NSCLC accountsfor 80-85% of all cases and is further divided into three major subtypesbased on histology: adenocarcinoma, squamous cell carcinoma (epidermoidcarcinoma), and large cell undifferentiated carcinoma.

Lung cancer typically presents late in its course, and thus has a mediansurvival of only 6-12 months after diagnosis and an overall 5 yearsurvival rate of only 5-10%. Although surgery offers the best chance ofa cure, only a small fraction of lung cancer patients are eligible withthe majority relying on chemotherapy and radiotherapy. Despite attemptsto manipulate the timing and dose intensity of these therapies, survivalrates have increased little over the last 15 years (Spiro et al., Am. J.Respir. Crit. Care Med. 166:1166-1196 (2002)).

Cancer arises from dysregulation of the mechanisms that control normaltissue development and maintenance, and increasingly stem cells arethought to play a central role (Beachy et al., Nature 432:324 (2004)).During normal animal development, cells of most or all tissues arederived from normal precursors, called stem cells (Morrison et al., Cell88:287-298 (1997); Morrison et al., Curr. Opin. Immunol. 9:216-221(1997); Morrison et al., Annu. Rev. Cell. Dev. Biol. 11:35-71 (1995)).Stem cells are cell that: (1) have extensive proliferative capacity; (2)are capable of asymmetric cell division to generate one or more kinds ofprogeny with reduced proliferative and/or developmental potential; and(3) are capable of symmetric cell divisions for self-renewal orself-maintenance. The best-known example of adult cell renewal by thedifferentiation of stem cells is the hematopoietic system wheredevelopmentally immature precursors (hematopoietic stem and progenitorcells) respond to molecular signals to form the varied blood andlymphoid cell types. Other cells, including cells of the gut, breastductal system, and skin are constantly replenished from a smallpopulation of stem cells in each tissue, and recent studies suggest thatmost other adult tissues also harbor stem cells, including the brain.

Solid tumors are composed of heterogeneous cell populations. Forexample, breast cancers are a mixture of cancer cells and normal cells,including mesenchymal (stromal) cells, inflammatory cells, andendothelial cells. Classic models of cancer hold that phenotypicallydistinct cancer cell populations all have the capacity to proliferateand give rise to a new tumor. In the classical model, tumor cellheterogeneity results from environmental factors as well as ongoingmutations within cancer cells resulting in a diverse population oftumorigenic cells. This model rests on the idea that all populations oftumor cells would have some degree of tumorigenic potential. (Pandis etal., Genes, Chromosomes & Cancer 12:122-129 (1998); Kuukasjrvi et al.,Cancer Res. 57.1597-1604 (1997); Bonsing et al., Cancer 71:382-391(1993); Bonsing et al., Genes Chromosomes & Cancer 82:173-183 (2000);Beerman H. et al., Cytometry. 12:147-154 (1991); Aubele M & Werner M,Analyt. Cell. Path. 19:53 (1999); Shen L. et al., Cancer Res. 60:3884(2000)).

An alternative model for the observed solid tumor cell heterogeneity isthat solid tumors result from a “solid tumor stem cell” (or “cancer stemcell” from a solid tumor) that subsequently undergoes chaoticdevelopment through both symmetric and asymmetric rounds of celldivisions. In this stem cell model, solid tumors contain a distinct andlimited (possibly even rare) subset of cells that share the propertiesof normal “stem cells”, in that they extensively proliferate andefficiently give rise both to additional solid tumor stem cells(self-renewal) and to the majority of tumor cells of a solid tumor thatlack tumorigenic potential. Indeed, mutations within a long-lived stemcell population may initiate the formation of cancer stem cells thatunderlie the growth and maintenance of tumors and whose presencecontributes to the failure of current therapeutic approaches.

The stem cell nature of cancer was first revealed in the blood cancer,acute myeloid leukemia (AML) (Lapidot et al., Nature 17:645-648 (1994)).More recently it has been demonstrated that malignant human breasttumors similarly harbor a small, distinct population of cancer stemcells enriched for the ability to form tumors in immunodeficient mice.An ESA+, CD44+, CD24−/low, Lin− cell population was found to be 50-foldenriched for tumorigenic cells compared to unfractionated tumor cells(Al-Hajj et al., PNAS 100:3983-3988 (2003)). The ability toprospectively isolate the tumorigenic cancer cells has permittedinvestigation of critical biological pathways that underlietumorigenicity in these cells, and thus promises the development ofbetter diagnostic assays and therapeutics for cancer patients. It istoward this purpose that this invention is directed.

BRIEF SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides a solublereceptor comprising a cancer stem cell marker. In certain embodiments,the soluble receptor comprises a cancer stem cell marker that binds aligand of the cancer stem cell marker. In certain embodiments, thesoluble receptor comprises a cancer stem cell marker and inhibits growthof tumor cells. In certain embodiments, the soluble receptor comprises aFri domain of a human FZD receptor. In certain embodiments, the solublereceptor comprises a Fri domain of a human FZD receptor that binds aligand of a human FZD receptor. In certain embodiments, the solublereceptor comprises a Fri domain of a human FZD receptor and inhibitsgrowth of tumor cells.

In certain embodiments, the present invention provides an isolatednucleic acid molecule that encodes a soluble receptor comprising acancer stem cell marker. In certain embodiments, the isolated nucleicacid molecule encodes a soluble receptor comprising a cancer stem cellmarker that binds a ligand of the cancer stem cell marker. In certainembodiments, the isolated nucleic acid molecule encodes a solublereceptor comprising a cancer stem cell marker that inhibits growth oftumor cells. In certain embodiments, the isolated nucleic acid moleculeencodes a soluble receptor comprising a Fri domain of a human FZDreceptor. In certain embodiments, the isolated nucleic acid moleculeencodes a soluble receptor comprising a Fri domain of a human FZDreceptor that binds a ligand of a human FZD receptor. In certainembodiments, the isolated nucleic acid molecule encodes a solublereceptor comprising a Fri domain of a human FZD receptor that inhibitsgrowth of tumor cells.

In certain embodiments, the present invention provides a method oftreating cancer, the method comprising administering a soluble receptorcomprising a cancer stem cell marker in an amount effective to inhibittumor cell growth. In certain embodiments the method of treating cancercomprises administering a soluble receptor comprising a cancer stem cellmarker that binds a ligand of the cancer stem cell marker in an amounteffective to inhibit tumor cell growth. In certain embodiments, themethod of treating cancer comprises administering a soluble receptorcomprising a Fri domain of a human FZD receptor in an amount effectiveto inhibit tumor cell growth. In certain embodiments, the method oftreating cancer comprises administering a soluble receptor comprising aFri domain of a human FZD receptor that binds a ligand of a human FZDreceptor in an amount effective to inhibit tumor cell growth.

Examples of solid tumors that can be treated using a therapeuticcomposition of the instant invention, for example, an antibody thatbinds a Fzd receptor or a receptor fusion protein that blocks ligandactivation of a Fzd receptor include, but are not limited to, sarcomasand carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. The invention is applicable to sarcomas and epithelialcancers, such as ovarian cancers and breast cancers.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: Half-life of FZD.Fc Soluble Receptors. Purified Fc fusionproteins were administered i.p. to 2 mice each and blood samples wereobtained at various times post-administration. FZD4 Fri.Fc, FZD5 Fri.Fc,and FZD8 Fri.Fc proteins are still present in the blood serum 72 hourspost-injection, and FZD5 Fri.Fc and FZD8 Fri.Fc proteins are present inthe blood serum up to 96 hours post-administration. In contrast, FZD5ECD.Fc is undetectable in blood serum after only 24 hours (top).

FIG. 2: FZD Fc Soluble Receptors Inhibit Wnt3a Signaling. Increasingconcentrations (2 nM, 5 nM, and 60 nM) of FZD Fc fusion proteinsincluding FZD4 Fri.Fc, FZD5 ECD.Fc, FZD5 Fri.Fc, and FZD8 Fri.Fc wereincubated with L cells in the presence or absence of Wnt3a ligand andthe stabilization of β-catenin was determined by immunoblotting. In theabsence of Wnt3a ligand, β-catenin could not be detected (LCM). In thepresence of Wnt3a β-catenin was stabilized, and this stabilization wasblocked by increasing amounts of FZD5, FZD8, and FZD4 Fc solublereceptor protein but not a control Fc protein (Con Fc).

FIG. 3: FZD Fc Soluble Receptors Inhibit Wnt Signaling. Hek 293 cellsstably transfected with 8×TCF-luciferase reporter were incubated withincreasing concentrations of FZD:Fc soluble receptors in the presence ofdifferent Wnt ligands including Wnt1, Wnt2, Wnt3, Wnt3a and Wnt7b. FZD4Fc, FZD5 Fc and FZD8 Fc fusion proteins inhibited Wnt signaling mediatedby all five Wnt ligands as shown by loss of luciferase activity.

FIG. 4: Reduction of Tumor Growth by FZDFc Soluble Receptor Proteins.NOD/SOD mice injected subcutaneously with dissociated colon tumor cells(10,000 cells per animal; n=10) were treated two days later withFZD7ECD.Fc soluble receptor, FZD10ECD.Fc soluble receptor, or controlinjections. Total tumor volume is shown for days 21, 24, 28 and 30. Thereduction of tumor volume by FZD7ECD.Fc was statistically significant onday 28 and day 30 (*).

FIG. 5: Prevention of Wnt-dependent Tumor Growth by FZD8 Fri.Fc SolubleReceptor Protein. NOD/SOD mice injected with 50,000 MMTV WNT1 tumorderived cells (n=10) were the following day treated with FZD8 Fri.Fcsoluble receptor or PBS as a control. Tumor growth was monitored weeklyuntil growth was detected, then tumor growth was measured twice a week.Tumor growth in animals treated with FZD Fri.Fc (left bar) was virtuallyeliminated compared to that observed in control animals (right bar).

FIG. 6: Reduction of PE13 Xenograft Tumor Growth by FZD8 Fri.Fc SolubleReceptor Protein. NOD/SCID mice injected with 50,000 PE13 breast tumorcells (n=10) were the following day treated with FZD8 Fri.Fc solublereceptor or PBS as a control. Tumor growth was monitored weekly untilgrowth was detected, then tumor growth was measured twice a week. Tumorgrowth in animals treated with FZD Fri.Fc (left bar) was significantlyreduced compared to that observed in control animals (right bar).

FIG. 7: Treatment of Wnt-dependent Tumor Growth by FZD Fri.Fc SolubleReceptor Protein. Female rag-2/γ chain double knockout mice wereimplanted with 50,000 MMTV Wnt1 breast tumor derived cells. Treatmentwith 5 mg/kg FZD8 Fri.Fc reduced the growth of tumors, as measured bytotal tumor volume over time, relative to mice treated with PBS (whitebars). Treatment with 10 mg/kg and 30 mg/kg FZD8 Fri.Fc was even moreeffective in reducing the size of the pre-established tumors. Incontrast, FZD5 Fri.Fc did not display anti-tumor effects on establishedbreast tumors that require wnt1 for growth.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “antagonist” is used herein to include any molecule thatpartially or fully blocks, inhibits, or neutralizes the expression of orthe biological activity of a cancer stem cell marker disclosed hereinand such biological activity includes, but is not limited to, inhibitionof tumor growth. The term “antagonist” includes any molecule thatpartially or fully blocks, inhibits, or neutralizes a biologicalactivity of the FZD pathway. Suitable antagonist molecules include, butare not limited to, fragments or amino acid sequence variants of nativeFZD receptors proteins including soluble FZD receptors.

The terms “isolated” and “purified” refer to material that issubstantially or essentially free from components that normallyaccompany it in its native state. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A protein(e.g. an soluble receptor) or nucleic acid that is the predominantspecies present in a preparation is substantially purified. Inparticular, an isolated nucleic acid is separated from open readingframes that naturally flank the gene and encode proteins other thanprotein encoded by the gene. An isolated antibody is separated fromother non-immunoglobulin proteins and from other immunoglobulin proteinswith different antigen binding specificity. It can also mean that thenucleic acid or protein is at least 85% pure, at least 95% pure, and insome embodiments at least 99% pure.

As used herein the terms “soluble receptor” and “FZD soluble receptor”refer to an N-terminal extracellular fragment of a human FZD receptorprotein preceding the first transmembrane domain of the receptor thatcan be secreted from a cell in soluble form. Both FZD soluble receptorscomprising the entire N-terminal extracellular domain (ECD) (referred toherein as “FZD ECD”) as well as smaller fragments are envisioned. FZDsoluble receptors comprising the Fri domain (referred to herein as “FZDFri”) are also disclosed. FZD Fri soluble receptors can demonstratealtered biological activity, (e.g. increased protein half-life) comparedto soluble receptors comprising the entire FZD ECD. Protein half-lifecan be further increased by covalent modification with poly(ethyleneglycol) or poly(ethylene oxide) (both referred to as PEG). FZD solublereceptors include FZD ECD or Fri domains fused in-frame to otherfunctional and structural proteins including, but not limited to, ahuman Fc (e.g. human Fc derived from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2,IgD, IgE, IgM); protein tags (e.g. myc, FLAG, GST); other endogenousproteins or protein fragments; or any other useful protein sequenceincluding any linker region between a FZD ECD or Fri domain and a linkedprotein. In certain embodiments the Fri domain of a FZD receptor islinked to human IgG1 Fc (referred to herein as “FZD Fri.Fc”). FZDsoluble receptors also include proteins with amino acid insertions,deletions, substitutions and conservative variations, etc.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

The terms “proliferative disorder” and “proliferative disease” refer todisorders associated with abnormal cell proliferation such as cancer.

“Tumor” and “neoplasm” as used herein refer to any mass of tissue thatresult from excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.

“Metastasis” as used herein refers to the process by which a cancerspreads or transfers from the site of origin to other regions of thebody with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

The terms “cancer stem cell”, “tumor stem cell”, or “solid tumor stemcell” are used interchangeably herein and refer to a population of cellsfrom a solid tumor that: (1) have extensive proliferative capacity; (2)are capable of asymmetric cell division to generate one or more kinds ofdifferentiated progeny with reduced proliferative or developmentalpotential; and (3) are capable of symmetric cell divisions forself-renewal or self-maintenance. These properties of “cancer stemcells”, “tumor stem cells” or “solid tumor stem cells” confer on thosecancer stem cells the ability to form palpable tumors upon serialtransplantation into an immunocompromised mouse compared to the majorityof tumor cells that fail to form tumors. Tumor cells, i.e.non-tumorigenic cells may form a tumor upon transplantation a limitednumber of times (e.g. one or two times) after obtaining the tumor cellsfrom a solid tumor but will not retain the capacity to form palpabletumors on serial transplantation into an immunocompromised mouse. Cancerstem cells undergo self-renewal versus differentiation in a chaoticmanner to form tumors with abnormal cell types that can change over timeas mutations occur. The solid tumor stem cells of the present inventiondiffer from the “cancer stem line” provided by U.S. Pat. No. 6,004,528.In that patent, the “cancer stem line” is defined as a slow growingprogenitor cell type that itself has few mutations but which undergoessymmetric rather than asymmetric cell divisions as a result oftumorigenic changes that occur in the cell's environment. This “cancerstem line” hypothesis thus proposes that highly mutated, rapidlyproliferating tumor cells arise largely as a result of an abnormalenvironment, which causes relatively normal stem cells to accumulate andthen undergo mutations that cause them to become tumor cells. U.S. Pat.No. 6,004,528 proposes that such a model can be used to enhance thediagnosis of cancer. The solid tumor stem cell model is fundamentallydifferent than the “cancer stem line” model and as a result exhibitsutilities not offered by the “cancer stem line” model. First, solidtumor stem cells are not “mutationally spared”. The “mutationally sparedcancer stem line” described by U.S. Pat. No. 6,004,528 can be considereda pre-cancerous lesion, while the solid tumor stem cells described bythis invention are cancer cells that themselves contain the mutationsthat are responsible for tumorigenesis. That is, the solid tumor stemcells (“cancer stem cells”) of the invention would be included among thehighly mutated cells that are distinguished from the “cancer stem line”in U.S. Pat. No. 6,004,528. Second, the genetic mutations that lead tocancer can be largely intrinsic within the solid tumor stem cells aswell as being environmental. The solid tumor stem cell model predictsthat isolated solid tumor stem cells can give rise to additional tumorsupon transplantation (thus explaining metastasis) while the “cancer stemline” model would predict that transplanted “cancer stem line” cellswould not be able to give rise to a new tumor, since it was theirabnormal environment that was tumorigenic. Indeed, the ability totransplant dissociated, and phenotypically isolated human solid tumorstem cells to mice (into an environment that is very different from thenormal tumor environment), where they still form new tumors,distinguishes the present invention from the “cancer stem line” model.Third, solid tumor stem cells likely divide both symmetrically andasymmetrically, such that symmetric cell division is not an obligateproperty. Fourth, solid tumor stem cells can divide rapidly or slowly,depending on many variables, such that a slow proliferation rate is nota defining characteristic.

The terms “cancer cell”, “tumor cell” and grammatical equivalents referto the total population of cells derived from a tumor including bothnon-tumorigenic cells, which comprise the bulk of the tumor cellpopulation, and tumorigenic stem cells also referred to herein as cancerstem cells.

As used herein “tumorigenic” refers to the functional features of asolid tumor stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells) that allow solid tumor stem cells to form atumor. These properties of self-renewal and proliferation to generateall other tumor cells confer on the cancer stem cells of this inventionthe ability to form palpable tumors upon serial transplantation into animmunocompromised mouse compared to the majority of tumor cells that areunable to form tumors upon the serial transplantation. Tumor cells, i.e.non-tumorigenic tumor cells, may form a tumor upon transplantation intoan immunocompromised mouse a limited number of times (for example one ortwo times) after obtaining the tumor cells from a solid tumor.

As used herein, the terms “stem cell cancer marker(s)”, “cancer stemcell marker(s)”, “tumor stem cell marker(s)”, or “solid tumor stem cellmarker(s)” refer to a gene or genes or a protein, polypeptide, orpeptide expressed by the gene or genes whose expression level, alone orin combination with other genes, is correlated with the presence oftumorigenic cancer cells compared to non-tumorigenic cells. Thecorrelation can relate to either an increased or decreased expression ofthe gene (e.g. increased or decreased levels of mRNA or the peptideencoded by the gene).

As used herein, the terms “unfractionated tumor cells”, “presorted tumorcells”, “bulk tumor cells”, and their grammatical equivalents are usedinterchangeably to refer to a tumor cell population isolated from apatient sample (e.g. a tumor biopsy or pleural effusion) that has notbeen segregated, or fractionated, based on cell surface markerexpression.

As used herein, the terms “non-ESA+CD44+ tumor cells”, “non-ESA+44+”,“sorted non-tumorigenic tumor cells”, “non-tumorigenic tumor cells,”“non-stem cells,” “tumor cells” and their grammatical equivalents areused interchangeably to refer to a tumor population from which thecancer stem cells of this invention have been segregated, or removed,based on cell surface marker expression.

As used herein, the terms “biopsy” and “biopsy tissue” refer to a sampleof tissue or fluid that is removed from a subject for the purpose ofdetermining if the sample contains cancerous tissue. In someembodiments, biopsy tissue or fluid is obtained because a subject issuspected of having cancer. The biopsy tissue or fluid is then examinedfor the presence or absence of cancer.

As used herein an “acceptable pharmaceutical carrier” refers to anymaterial that, when combined with an active ingredient of apharmaceutical composition such as an antibody, allows the antibody, forexample, to retain its biological activity. In addition, an “acceptablepharmaceutical carrier” does not trigger an immune response in arecipient subject. Examples include, but are not limited to, any of thestandard pharmaceutical carriers such as a phosphate buffered salinesolution, water, and various oil/water emulsions. Examples of diluentsfor aerosol or parenteral administration are phosphate buffered salineor normal (0.9%) saline.

The term “therapeutically effective amount” refers to an amount of asoluble receptor, or other drug effective to “treat” a disease ordisorder in a subject or mammal. In the case of cancer, thetherapeutically effective amount of the drug can reduce the number ofcancer cells; reduce the tumor size; inhibit or stop cancer cellinfiltration into peripheral organs; inhibit or stop tumor metastasis;inhibit and stop tumor growth; and/or relieve to some extent one or moreof the symptoms associated with the cancer. To the extent the drugprevents growth and/or kills existing cancer cells, it can be referredto as cytostatic and/or cytotoxic.

As used herein the term “inhibit tumor growth” refers to any mechanismby which tumor cell growth can be inhibited. In certain embodimentstumor cell growth is inhibited by slowing proliferation of tumor cells.In certain embodiments tumor cell growth is inhibited by haltingproliferation of tumor cells. In certain embodiments tumor cell growthis inhibited by killing tumor cells. In certain embodiments tumor cellgrowth is inhibited by inducing apoptosis of tumor cells. In certainembodiments tumor cell growth is inhibited by depriving tumor cells ofnutrients. In certain embodiments tumor cell growth is inhibited bypreventing migration of tumor cells. In certain embodiments tumor cellgrowth is inhibited by preventing invasion of tumor cells.

As used herein, “providing a diagnosis” or “diagnostic information”refers to any information that is useful in determining whether apatient has a disease or condition and/or in classifying the disease orcondition into a phenotypic category or any category having significancewith regards to the prognosis of or likely response to treatment (eithertreatment in general or any particular treatment) of the disease orcondition. Similarly, diagnosis refers to providing any type ofdiagnostic information, including, but not limited to, whether a subjectis likely to have a condition (such as a tumor), information related tothe nature or classification of a tumor as for example a high risk tumoror a low risk tumor, information related to prognosis and/or informationuseful in selecting an appropriate treatment. Selection of treatment caninclude the choice of a particular chemotherapeutic agent or othertreatment modality such as surgery or radiation or a choice aboutwhether to withhold or deliver therapy.

As used herein, the terms “providing a prognosis”, “prognosticinformation”, or “predictive information” refer to providing informationregarding the impact of the presence of cancer (e.g., as determined bythe diagnostic methods of the present invention) on a subject's futurehealth (e.g., expected morbidity or mortality, the likelihood of gettingcancer, and the risk of metastasis).

Terms such as “treating”, “treatment”, “to treat”, “alleviating”, and“to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent or slow the development of a targeted pathologiccondition or disorder. Thus those in need of treatment include thosealready with the disorder; those prone to have the disorder; and thosein whom the disorder is to be prevented. A subject is successfully“treated” according to the methods of the present invention if thepatient shows one or more of the following: a reduction in the number ofor complete absence of cancer cells; a reduction in the tumor size;inhibition of or an absence of cancer cell infiltration into peripheralorgans including the spread of cancer into soft tissue and bone;inhibition of or an absence of tumor metastasis; inhibition or anabsence of tumor growth; relief of one or more symptoms associated withthe specific cancer; reduced morbidity and mortality; and improvement inquality of life.

As used herein, the terms “polynucleotide” and “nucleic acid” refer to apolymer composed of a multiplicity of nucleotide units (ribonucleotideor deoxyribonucleotide or related structural variants) linked viaphosphodiester bonds, including but not limited to, DNA or RNA. The teenencompasses sequences that include any of the known base analogs of DNAand RNA including, but not limited to, 4 acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5(carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5 bromouracil,5-carboxymethylaminomethyl 2 thiouracil, 5 carboxymethyl

aminomethyluracil, dihydrouracil, inosine, N6 isopentenyladenine, 1methyladenine, 1-methylpseudo

uracil, 1 methylguanine, 1 methyl inosine, 2,2-dimethyl

guanine, 2 methyladenine, 2 methylguanine, 3-methyl

cytosine, 5 methylcytosine, N6 methyladenine, 7 methylguanine, 5methylaminomethyluracil, 5-methoxy

amino

methyl 2 thiouracil, beta D mannosylqueosine, 5′methoxycarbonylmethyluracil, 5 methoxyuracil, 2 methylthio N6isopentenyladenine, uracil 5 oxyacetic acid methylester, uracil 5oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2 thiocytosine,5-methyl-2 thiouracil, 2-thiouracil, 4 thiouracil, 5-methyluracil,N-uracil 5 oxyacetic acid methylester, uracil 5 oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, and 2,6 diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) molecule thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns cancontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide. In addition to containing introns, genomic forms ofa gene can also include sequences located on both the 5′ and 3′ end ofthe sequences that are present on the RNA transcript. These sequencesare referred to as “flanking” sequences or regions (these flankingsequences are located 5′ or 3′ to the non-translated sequences presenton the mRNA transcript). The 5′ flanking region can contain regulatorysequences such as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region can contain sequencesthat direct the termination of transcription, post transcriptionalcleavage and polyadenylation.

The term “recombinant” when used with reference to a cell, nucleic acid,protein or vector indicates that the cell, nucleic acid, protein orvector has been modified by the introduction of a heterologous nucleicacid or protein, the alteration of a native nucleic acid or protein, orthat the cell is derived from a cell so modified. Thus, e.g.,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell or express native genes that areoverexpressed or otherwise abnormally expressed such as, for example,expressed as non-naturally occurring fragments or splice variants. Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid, e.g., using polymerases and endonucleases, in a form not normallyfound in nature. In this manner, operably linkage of different sequencesis achieved. Thus an isolated nucleic acid molecule, in a linear form,or an expression vector formed in vitro by ligating DNA molecules thatare not normally joined, are both considered recombinant for thepurposes of this invention. It is understood that once a recombinantnucleic acid molecule is made and introduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid molecule as depicted above.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” Vectorsare often derived from plasmids, bacteriophages, or plant or animalviruses.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments. Unless otherwise provided,ligation can be accomplished using known buffers and conditions with 10units to T4 DNA ligase (“ligase”) per 0.5 ug of approximately equimolaramounts of the DNA fragments to be ligated. Ligation of nucleic acid canserve to link two proteins together in-frame to produce a singleprotein, or fusion protein.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (e.g., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (e.g., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

The terms “polypeptide,” “peptide,” “protein”, and “protein fragment”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refersto compounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an alpha carbon that is bound to a hydrogen,a carboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs can have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. “Amino acid variants” refers to amino acidsequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical or associated (e.g., naturally contiguous) sequences. Becauseof the degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode most proteins. For instance, the codonsGCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at everyposition where an alanine is specified by a codon, the codon can bealtered to another of the corresponding codons described withoutaltering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes silent variations of the nucleic acid. One ofordinary skill will recognize that in certain contexts each codon in anucleic acid (except AUG, which is ordinarily the only codon formethionine, and TGG, which is ordinarily the only codon for tryptophan)can be modified to yield a functionally identical molecule. Accordingly,silent variations of a nucleic acid which encodes a polypeptide isimplicit in a described sequence with respect to the expression product,but not with respect to actual probe sequences. As to amino acidsequences, one of skill will recognize that individual substitutions,deletions or additions to a nucleic acid, peptide, polypeptide, orprotein sequence which alters, adds or deletes a single amino acid or asmall percentage of amino acids in the encoded sequence is a“conservatively modified variant” including where the alteration resultsin the substitution of an amino acid with a chemically similar aminoacid. Conservative substitution tables providing functionally similaramino acids are well known in the art. Such conservatively modifiedvariants are in addition to and do not exclude polymorphic variants,interspecies homologs, and alleles of the invention. Typicallyconservative substitutions include: 1) Alanine (A), Glycine (G); 2)Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton, Proteins (1984)).

The term “epitope tagged” as used herein refers to a chimericpolypeptide comprising a cancer stem cell marker protein, or a domainsequence or portion thereof, fused to an “epitope tag”. The epitope tagpolypeptide comprises enough amino acid residues to provide an epitopefor recognition by an antibody, yet is short enough such that it doesnot interfere with the activity of the cancer stem cell marker protein.Suitable epitope tags generally have at least six amino acid residues,usually between about 8 to about 50 amino acid residues, or about 10 toabout 20 residues. Commonly used epitope tags include Fc, HA, His, andFLAG tags.

As used herein, “about” refers to plus or minus 10% of the indicatednumber. For example, “about 10%” indicates a range of 9% to 11%.

Detailed Description

The present invention provides compositions and methods for studying,diagnosing, characterizing, and treating cancer. In particular, thepresent invention provides antagonists against solid tumor stem cellmarkers and methods of using these antagonists to inhibit tumor growthand treat cancer in human patients. Antagonists include soluble receptorproteins comprising cancer stem cell markers. In certain embodiments,the present invention provides a soluble receptor comprising a Fridomain of a human FZD receptor that inhibits growth of tumor cells. Incertain embodiments, the soluble receptor comprises the Fri domain ofhuman FZD4. In certain embodiments, the soluble receptor comprises theFri domain of human FZD4 comprising the amino acid sequence of SEQ IDNO: 8. In certain embodiments, the soluble receptor comprises the Fridomain of human FZD4 linked in-frame to a non-FZD receptor proteinsequence. In certain embodiments, the soluble receptor comprises the Fridomain of human FZD4 linked in-frame to human Fc. In certainembodiments, the soluble receptor comprises the Fri domain of human FZD4linked in-frame to human IgG₁ Fc. In certain embodiments, the solublereceptor comprises the Fri domain of human FZD4 linked in-frame to humanIgG₁ Fc comprising an amino acid sequence shown in SEQ ID NO: 4.

In certain embodiments, the soluble receptor comprises the Fri domain ofhuman FZD5. In certain embodiments, the soluble receptor comprises theFri domain of human FZD5 comprising the amino acid sequence of SEQ IDNO: 9. In certain embodiments, the soluble receptor comprises the Fridomain of human FZD5 linked in-frame to a non-FZD receptor proteinsequence. In certain embodiments, the soluble receptor comprises the Fridomain of human FZD5 linked in-frame to human Fc. In certainembodiments, the soluble receptor comprises the Fri domain of human FZD5linked in-frame to human IgG₁ Fc. In certain embodiments, the solublereceptor comprises the Fri domain of human FZD5 linked in-frame to humanIgG₁ Fc comprising an amino acid sequence shown in SEQ ID NO: 4.

In certain embodiments, the soluble receptor comprises the Fri domain ofhuman FZD8. In certain embodiments, the soluble receptor comprises theFri domain of human FZD8 comprising the amino acid sequence of SEQ IDNO: 7. In certain embodiments, the soluble receptor comprises the Fridomain of human FZD8 linked in-frame to a non-FZD receptor proteinsequence. In certain embodiments, the soluble receptor comprises the Fridomain of human FZD8 linked in-frame to human Fc. In certainembodiments, the soluble receptor comprises the Fri domain of human FZD8linked in-frame to human IgG₁ Fc. In certain embodiments, the solublereceptor comprises the Fri domain of human FZD8 linked in-frame to humanIgG₁ Fc comprising an amino acid sequence shown in SEQ ID NO: 4.

In certain embodiments, the present invention provides an isolatednucleic acid encoding a soluble receptor comprising: a nucleic acidsequence encoding a Fri domain of human FZD4 comprising an amino acidsequence shown in SEQ ID NO: 8; and a nucleic acid sequence encodinghuman IgG₁ Fc comprising an amino acid sequence shown in SEQ ID NO: 4.In certain embodiment, the present invention provides a vectorcomprising the nucleic acid sequence encoding a Fri domain of human FZD4comprising an amino acid sequence shown in SEQ ID NO: 8; and the nucleicacid sequence encoding human IgG₁ Fc comprising an amino acid sequenceshown in SEQ ID NO: 4, In certain embodiments the vector is operablylinked to control sequences recognized by a host cell transformed withthe vector. In certain embodiments, the present invention provides anisolated host cell comprising the vector comprising the nucleic acidsequence encoding a Fri domain of human FZD4 comprising an amino acidsequence shown in SEQ ID NO: 8; and the nucleic acid sequence encodinghuman IgG₁ Fc comprising an amino acid sequence shown in SEQ ID NO: 4.

In certain embodiments, the present invention provides an isolatednucleic acid molecule encoding a soluble receptor comprising: a nucleicacid sequence encoding a Fri domain of human FZD5 comprising an aminoacid sequence shown in SEQ ID NO: 9; and a nucleic acid sequenceencoding human IgG₁ Fc comprising an amino acid sequence shown in SEQ IDNO: 4. In certain embodiments, the present invention provides a vectorcomprising the nucleic acid sequence encoding a Fri domain of human FZD5comprising an amino acid sequence shown in SEQ ID NO: 9; and the nucleicacid sequence encoding human IgG₁ Fc comprising an amino acid sequenceshown in SEQ ID NO: 4. In certain embodiments the vector is operablylinked to control sequences recognized by a host cell transformed withthe vector. In certain embodiments, the present invention provides anisolated host cell comprising the vector comprising the nucleic acidsequence encoding a Fri domain of human FZD5 comprising an amino acidsequence shown in SEQ ID NO: 9; and the nucleic acid sequence encodinghuman IgG₁ Fc comprising an amino acid sequence shown in SEQ ID NO: 4.

In certain embodiments, the present invention provides an isolatednucleic acid molecule encoding a soluble receptor comprising: a nucleicacid sequence encoding a Fri domain of human FZD8 comprising an aminoacid sequence shown in SEQ ID NO: 7; and a nucleic acid sequenceencoding human IgG₁ Fc comprising an amino acid sequence shown in SEQ IDNO: 4. In certain embodiment, the present invention provides a vectorcomprising the nucleic acid sequence encoding a Fri domain of human FZD8comprising an amino acid sequence shown in SEQ ID NO: 7; and the nucleicacid sequence encoding human IgG₁ Fc comprising an amino acid sequenceshown in SEQ ID NO: 4. In certain embodiments the vector is operablylinked to control sequences recognized by a host cell transformed withthe vector. In certain embodiments, the present invention provides anisolated host cell comprising the vector comprising the nucleic acidsequence encoding a Fri domain of human FZD8 comprising an amino acidsequence shown in SEQ ID NO: 7; and the nucleic acid sequence encodinghuman IgG₁ Fc comprising an amino acid sequence shown in SEQ ID NO: 4.

In certain embodiments, the present invention provides a pharmaceuticalcomposition comprising a soluble receptor. In certain embodiments, thepharmaceutical composition comprises a soluble receptor comprising theFri domain of a human FZD receptor. In certain embodiments thepharmaceutical composition comprises a soluble receptor comprising theFri domain of human FZD4 receptor. In certain embodiments thepharmaceutical composition comprises a soluble receptor comprising theFri domain of human FZD5 receptor. In certain embodiments, thepharmaceutical composition comprises a soluble receptor comprising theFri domain of human FZD8 receptor.

In certain embodiments, the present invention provides a method oftreating cancer comprising administering a soluble receptor comprising aFri domain of a human FZD receptor in an amount effective to inhibittumor cell growth. In certain embodiments a method of treating cancercomprises administering a soluble receptor comprising a Fri domain ofhuman FZD4 receptor in an amount effective to inhibit tumor cell growth.In certain embodiments a method of treating cancer comprisesadministering a soluble receptor comprising a Fri domain of human FZD5receptor in an amount effective to inhibit tumor cell growth. In certainembodiments a method of treating cancer comprises administering asoluble receptor comprising a Fri domain of human FZD8 receptor in anamount effective to inhibit tumor cell growth.

In certain embodiments the method of treating cancer comprisesadministering a soluble receptor comprising the Fri domain of a humanFZD receptor linked in-frame to a non-FZD receptor protein sequence inan amount effective to inhibit tumor cell growth. In certain embodimentsthe method of treating cancer comprises administering a soluble receptorcomprising the Fri domain of a human FZD receptor linked in-frame to ahuman Fc in an amount effective to inhibit tumor cell growth. In certainembodiments the method of treating cancer comprises administering asoluble receptor comprising the Fri domain of a human FZD receptorlinked in-frame to human IgG₁ Fc in an amount effective to inhibit tumorcell growth. In certain embodiments the method of treating cancercomprises administering a soluble receptor comprising the Fri domain ofa human FZD receptor linked in-frame to human IgG₁ Fc comprising asamino acid sequence shown in SEQ ID NO: 4 in an amount effective toinhibit tumor cell growth.

In certain embodiments, the present invention provides a method oftreating cancer comprises administering a soluble receptor comprisingthe Fri domain of a human FZD receptor in an amount effective to inhibittumor cell growth in combination with radiation therapy. In certainembodiments the method of treating cancer comprises administering asoluble receptor comprising the Fri domain of a human FZD receptor in anamount effective to inhibit tumor cell growth in combination withchemotherapy. In certain embodiments the method of treating cancercomprising administering a soluble receptor comprising the Fri domain ofa human FZD receptor in an amount effective to inhibit tumor cell growthof tumor cells from a breast tumor, colorectal tumor, lung tumor,pancreatic tumor, prostate tumor, or a head and neck tumor.

Stem Cells and Solid Tumor Stem Cells

Common cancers arise in tissues that contain a subpopulation ofproliferating cells that are responsible for replenishing theshort-lived mature cells. In such organs, cell maturation is arranged ina hierarchy in which a rare population of stem cells give rise both tothe more differentiated cells and perpetuate themselves through aprocess called self renewal (Akashi & Weissman, Developmental Biology ofHematopoiesis, Oxford Univ. Press, NY (2001); Spangrude et al., Science241:58-61 (1988); Baum et al., PNAS 89:2804-2808 (1992); Morrison etal., PNAS 92:10302-20306 (1995); Morrison et al., Immunity 5:207-216(1996); Morrison et al., Annu. Rev. Cell Dev. Biol. 11:35-71 (1995);Morrison et al., Dev. 124:1929-1939 (1997); Morrison & Weissman,Immunity 1:661 (1994); Morrison et al., Cell 88:287-298 (1997); Uchidaet al., PNAS 97: 14720-14725 (2000); Morrison et al., Cell 101:499-510(2000)). Although it is likely that most tissues contain stem cells, dueto their rarity these cells have been rigorously identified and purifiedto study their biological, molecular, and biochemical properties in onlya few tissues. The best characterized stem cells are those that giverise to the hematopoietic system, called hematopoietic stem cells(HSCs). The utility of HSCs has been demonstrated in cancer therapy withtheir extensive use for bone marrow transplantation to regenerate thehematolymphoid system following myeloablative protocols (Baum et al.,Bone Marrow Transplantation, Blackwell Scientific Publications, Boston(1994)). Understanding the cellular biology of the tissues in whichcancers arise, and specifically of the stem cells residing in thosetissues, promises to provide new insights into cancer biology.

Like the tissues in which they originate, solid tumors consist of aheterogeneous population of cells. That the majority of these cells lacktumorigenicity suggested that the development and maintenance of solidtumors also relies on a small population of stem cells (i.e.,tumorigenic cancer cells) with the capacity to proliferate andefficiently give rise both to additional tumor stem cells (self-renewal)and to the majority of more differentiated tumor cells that lacktumorigenic potential (i.e., non-tumorigenic cancer cells). The conceptof cancer stem cells was first introduced soon after the discovery ofHSC and was established experimentally in acute myelogenous leukemia(AML) (Park et al., J. Natl. Cancer Inst. 46:411-422 (1971); Lapidot etal., Nature 367:645-648 (1994); Bonnet & Dick, Nat. Med. 3:730-737(1997); Hope et al., Nat. Immunol. 5:738-743 (2004)). Stem cells fromsolid tumors have more recently been isolated based on their expressionof a unique pattern of cell-surface receptors and on the assessment oftheir properties of self-renewal and proliferation in culture and inxenograft animal models. An ESA+ CD44+ CD24−/low Lineage− populationgreater than 50-fold enriched for the ability to form tumors relative tounfractionated tumor cells was discovered (Al-Hajj et al., PNAS100:3983-3988 (2003)). The ability to isolate tumorigenic cancer stemcells from the bulk of non-tumorigenic tumor cells has led to theidentification of cancer stem cell markers, genes with differentialexpression in cancer stem cells compared to non-tumorigenic tumor cellsor normal breast epithelium, using microarray analysis. The presentinvention employs the knowledge of these identified cancer stem cellmarkers to study, characterize, diagnosis and treat cancer.

Cancer Stem Cell Marker Protein

Normal stem cells and cancer stem cells share the ability to proliferateand self-renew, thus is not surprising that a number of genes thatregulate normal stem cell development contribute to tumorigenesis(reviewed in Reya et al., Nature 414:105-111 (2001) and Taipale &Beachy, Nature 411:349-354 (2001)). The present invention identifies Fzdreceptor, including for example, Fzd4, Fzd5, and Fzd8 as markers ofcancer stem cells, implicating the Wnt signaling pathway in themaintenance of cancer stem cells and as a target for treating cancer viathe elimination of these tumorigenic cells.

The Wnt signaling pathway is one of several critical regulators ofembryonic pattern formation, post-embryonic tissue maintenance, and stemcell biology. More specifically, Wnt signaling plays an important rolein the generation of cell polarity and cell fate specification includingself-renewal by stem cell populations. Unregulated activation of the Wntpathway is associated with numerous human cancers where it can alter thedevelopmental fate of tumor cells to maintain them in anundifferentiated and proliferative state. Thus carcinogenesis canproceed by usurping homeostatic mechanisms controlling normaldevelopment and tissue repair by stem cells (reviewed in Reya & Clevers,Nature 434:843 (2005); Beachy et al., Nature 432:324 (2004)).

The Wnt signaling pathway was first elucidated in the Drosophiladevelopmental mutant wingless (wg) and from the murine proto-oncogeneint-1, now Wnt1 (Nusse & Varmus, Cell 31:99-109 (1982); Van Ooyen &Nusse, Cell 39:233-240 (1984); Cabrera et al., Cell 50:659-663 (1987);Rijsewijk et al., Cell 50:649-657 (1987)). Wnt genes encode secretedlipid-modified glycoproteins of which 19 have been identified inmammals. These secreted ligands activate a receptor complex consistingof a Frizzled (Fzd) receptor family member and low-density lipoprotein(LDL) receptor-related protein 5 or 6 (LPR5/6). The Fzd receptors areseven transmembrane domain proteins of the G-protein coupled receptor(GPCR) superfamily and contain a large extracellular N-terminal ligandbinding domain with 10 conserved cysteines, known as a cysteine-richdomain (CRD) or Fri domain. There are ten human FZD receptors: FZD1-10.Different Fzd CRDs have different binding affinities for specific Wnts(Wu & Nusse, J. Biol. Chem. 277:41762-41769 (2002)), and Fzd receptorshave been grouped into those that activate the canonical β-cateninpathway and those that activate non-canonical pathways described below(Miller et al., Oncogene 18:7860-7872 (1999)). LRP5/6 are single passtransmembrane proteins with four extracellular EGF-like domainsseparated by six YWTD amino acid repeats that contribute to Fzd andligand binding (Johnson et al., J Bone Mineral Res 19:1749 (2004)).

The canonical Wnt signaling pathway activated upon receptor binding ismediated by the cytoplasmic protein Dishevelled (Dsh) interactingdirectly with the Fzd receptor and results in the cytoplasmicstabilization and accumulation of β-catenin. In the absence of a Wntsignal, β-catenin is localized to a cytoplasmic destruction complex thatincludes the tumor suppressor proteins adenomatous polyposis coli (APC)and auxin. These proteins function as critical scaffolds to allowglycogen synthase kinase (GSK)-3β to bind and phosphorylate β-catenin,marking it for degradation via the ubiquitin/proteasome pathway.Activation of Dsh results in phosphorylation of GSK3β and thedissociation of the destruction complex. Accumulated cytoplasmicβ-catenin is then transported into the nucleus where it interacts withthe DNA-binding proteins of the Tcf/Lef family to activatetranscription.

In addition to the canonical signaling pathway, Wnt ligands alsoactivate β-catenin-independent pathways (Veeman et al., Dev. Cell5:367-377 (2003)). Non-canonical Wnt signaling has been implicated innumerous processes but most convincingly in gastrulation movements via amechanism similar to the Drosophila planar cell polarity (PCP) pathway.Other potential mechanisms of non-canonical Wnt signaling includecalcium flux, JNK, and both small and heterotrimeric G-proteins.Antagonism is often observed between the canonical and non-canonicalpathways, and some evidence indicates that non-canonical signaling cansuppress cancer formation (Olson & Gibo, Exp. Cell Res. 241:134 (1998);Topol et al., J. Cell Biol. 162:899-908 (2003)).

Hematopoietic stem cells (HSCs) are the best understood stem cells inthe body, and Wnt signaling is implicated both in their normalmaintenance as well as in leukemic transformation (Reya & Clevers, 2005,Nature 434:843). HSCs are a rare population of cells that reside in astomal niche within the adult bone marrow. These cells are characterizedboth by a unique gene expression profile as well as an ability tocontinuously give rise to more differentiated progenitor cells toreconstitute the entire hematopoietic system. Both HSCs and the cells oftheir stromal microenvironment express Wnt ligands, and Wnt reporteractivation is present in HSCs in vivo. Furthermore, both β-catenin andpurified Wnt3A promote self-renewal of murine HSCs in vitro and enhancetheir ability to reconstitute the hematopoietic system in vivo whileWnt5A promotes expansion of human hematopoietic progenitors in vitro andre-population in a NOD-SCID xenotransplant model (Reya et al., Nature423:409-414 (2003); Willert et al., Nature 423:448-452 (2003); Van DenBerg et al., Blood 92:3189-3202 (1998); Murdoch et al., PNAS100:3422-3427 (2003)).

More recently Wnt signaling has been found to play a role in theoncogenic growth of both myeloid and lymphoid lineages. For example,granulocyte-macrophage progenitors (GMPs) from chronic myelogenousleukemias display activated Wnt signaling on which they are depended forgrowth and renewal (Jamieson et al., N. Engl. J. Med. 351:657-667(2004)) And while leukemias do not appear to harbor mutations within theWnt pathway, autocrine and/or paracrine Wnt signaling can sustaincancerous self-renewal (Reya & Clevers, Nature 434:843 (2005)).

The canonical Wnt signaling pathway also plays a central role in themaintenance of stem cell populations in the small intestine and colon,and the inappropriate activation of this pathway plays a prominent rolein colorectal cancers (Reya & Clevers, Nature 434:843 (2005)). Theabsorptive epithelium of the intestines is arranged into villi andcrypts. Stem cells reside in the crypts and slowly divide to producerapidly proliferating cells which give rise to all the differentiatedcell populations that move up out of the crypts to occupy the intestinalvilli. The Wnt signaling cascade plays a dominant role in controllingcell fates along the crypt-villi axis and is essential for themaintenance of the stem cell population. Disruption of Wnt signalingeither by genetic loss of Tcf7/2 by homologous recombination (Korinek etal., Nat. Genet. 19:379 (1998)) or overexpression of Dickkopf-1 (Dkk1),a potent secreted Wnt antagonist (Pinto et al., Genes Dev. 17:1709-1713(2003); Kuhnert et al., PNAS 101:266-271 (2004)), results in depletionof intestinal stem cell populations.

Colorectal cancer is most commonly initiated by activating mutations inthe Wnt signaling cascade. Approximately 5-10% of all colorectal cancersare hereditary with one of the main forms being familial adenomatouspolyposis (FAP), an autosomal dominant disease in which about 80% ofaffected individuals contain a germline mutation in the adenomatouspolyposis coli (APC) gene. Mutations have also been identified in otherWnt pathway components including auxin and β-catenin. Individualadenomas are clonal outgrowths of epithelial cell containing a secondinactivated allele, and the large number of FAP adenomas inevitablyresults in the development of adenocarcinomas through addition mutationsin oncogenes and/or tumor suppressor genes. Furthermore, activation ofthe Wnt signaling pathway, including gain-of-function mutations in APCand β-catenin, can induce hyperplastic development and tumor growth inmouse models (Oshima et al., Cancer Res. 57:1644-1649 (1997); Harada etal., EMBO J. 18:5931-5942 (1999)).

A role for Wnt signaling in cancer was first uncovered with theidentification of Wnt1 (originally int1) as an oncogene in mammarytumors transformed by the nearby insertion of a murine virus (Nusse &Varmus, Cell 31:99-109 (1982)). Additional evidence for the role of Wntsignaling in breast cancer has since accumulated. For instance,transgenic overexpression of β-catenin in the mammary glands results inhyperplasias and adenocarcinomas (Imbert et al., J. Cell Biol.153:555-568 (2001); Michaelson & Leder, Oncogene 20:5093-5099 (2001))whereas loss of Wnt signaling disrupts normal mammary gland development(Tepera et al., J. Cell Sc. 116:1137-1140 (2003); Hatsell et al., J.Mammary Gland Biol. Neoplasia 8:145-158 (2003)). More recently mammarystem cells have been shown to be activated by Wnt signaling (Liu et al.,PNAS 101:4158 (2004)). In human breast cancer, β-catenin accumulationimplicates activated Wnt signaling in over 50% of carcinomas, and thoughspecific mutations have not been identified, upregulation of Frizzledreceptor expression has been observed (Brennan & Brown, J. Mammary GlandNeoplasia 9:119-131 (2004); Malovanovic et al., Int. J. Oncol.25:1337-1342 (2004)).

FZD10, FZD8, FZD7, FZD4, and FZD5 are five of ten identified human Wntreceptors. In the mouse embryo Fzd10 is expressed with Wnt7a in theneural tube, limb buds, and Mullerian duct (Nunnally & Parr, Dev. GenesEvol. 214:144-148 (2004)) and can act as a receptor for Wnt-7a duringlimb bud development (Kawakami et al., Dev. Growth Differ. 42:561-569(2000)). Fzd10 is co-expressed with Wnt7b in the lungs, and celltransfection studies have demonstrated that the Fzd10/LRP5 co-receptoractivates the canonical Wnt signaling pathway in response to Wnt7b (Wanget al., Mol. Cell Biol. 25:5022-5030 (2005)). FZD10 mRNA is upregulatedin numerous cancer cell lines, including cervical, gastric, andglioblastoma cell lines, and in primary cancers including approximately40% of primary gastric cancers, colon cancers, and synovial sarcomas(Saitoh et al., Int. J. Oncol. 20:117-120 (2002); Terasaki et al., Int.J. Mol, Med. 9:107-112 (2002); Nagayama et al., Oncogene 1-12 (2005)).FZD8 is upregulated in several human cancer cell lines, primary gastriccancers, and renal carcinomas (Saitoh et al., Int. J. Oncol. 18:991-996(2001); Kirikoshi et al., Int. J. Oncol. 19:111-115 (2001); Janssens etal., Tumor Biol. 25:161-171 (2004)). FZD7 is expressed throughout thegastrointestinal tract and is up-regulated in one out of six cases ofhuman primary gastric cancer (Kirikoshi et al., Int. J. Oncol.19:111-115 (2001)). Expression of the FZD7 ectodomain by a colon cancercell line induced morphological changes and decreased tumor growth in axenograft model (Vincan et al., Differentiation 73:142-153 (2005)). FZD5plays an essential role in yolk sac and placental angiogenesis (Ishikawaet al., Dev. 128:25-33 (2001)) and is upregulated in renal carcinomas inassociation with activation of Wnt/β-catenin signaling (Janssens et al.,Tumor Biology 25:161-171 (2004)). FZD4 is highly expressed in intestinalcrypt epithelial cells and is one of several factors that displaydifferential expression in normal versus neoplastic tissue (Gregorieffet al., Gastroenterology 129:626-638 (2005)). The identification ofFZD4, 5, 7, 8, and 10 as markers of cancer stem cells thus makes theseproteins ideal targets for cancer therapeutics.

Diagnostic Assays

The present invention provides a cancer stem cell marker the expressionof which can be analyzed to detect, characterize, diagnosis or monitor adisease associated with expression of a cancer stem cell marker. Incertain embodiments, expression of a cancer stem cell marker isdetermined by polynucleotide expression such as, for example, mRNAencoding the cancer stem cell marker. The polynucleotide can be detectedand quantified by any of a number of means well known to those of skillin the art. In some embodiments, mRNA encoding a cancer stem cell markeris detected by in situ hybridization of tissue sections from, fromexample, a patient biopsy. Alternatively, RNA can be isolated from atissue and detected by, for example, Northern blot, quantitative RT-PCRor microarrays. For example, total RNA can be extracted from a tissuesample and primers that specifically hybridize and amplify a cancer stemcell marker can be used to detect expression of a cancer stem cellmarker polynucleotide using RT-PCR.

In certain embodiments, expression of a cancer stem cell marker can bedetermined by detection of the corresponding polypeptide. Thepolypeptide can be detected and quantified by any of a number of meanswell known to those of skill in the art. In some embodiments, a cancerstem cell marker polypeptide is detected using analytic biochemicalmethods such as, for example, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC) or thinlayer chromatography (TLC). The isolated polypeptide can also besequenced according to standard techniques. In some embodiments, acancer stem cell marker protein is detected with antibodies raisedagainst the protein using, for example, immunofluorescence orimmunohistochemistry on tissue sections. Alternatively antibodiesagainst a cancer stem cell marker can detect expression using, forexample, ELISA, FACS, Western blot, immunoprecipitation or proteinmicroarrays. For example, cancer stem cells can be isolated from apatient biopsy and expression of a cancer stem cell marker proteindetected with fluorescently labeled antibodies using FACS. In anothermethod, the cells expressing a cancer stem cell marker can be detectedin vivo using labeled antibodies typical imaging system. For example,antibodies labeled with paramagnetic isotopes can be used for magneticresonance imaging (MRI).

In some embodiments of the present invention, a diagnostic assaycomprises determining the expression or not of a cancer stem cell markerin tumor cells using, for example, immunohistochemistry, in situhybridization, or RT-PCR. In other embodiments, a diagnostic assaycomprises determining expression levels of a cancer stem cell markerusing, for example, quantitative RT-PCR. In some embodiments, adiagnostic assay further comprises determining expression levels of acancer stem cell marker compared to a control tissue such as, forexample, normal epithelium.

Detection of a cancer stem cell marker expression can then be used toprovide a prognosis. A prognosis can be based on any known riskexpression of a cancer stem cell marker can indicate. Furthermore,detection of a cancer stem cell marker can be used to select anappropriate therapy including, for example, treatment with an antagonistagainst the detected cancer stem cell marker. In some embodiments, theantagonist is an antibody that specifically binds to the extracellulardomain of a cancer stem cell marker protein.

Cancer Stem Cell Marker Antagonists

In the context of the present invention, a suitable antagonist is anagent that can have one or more of the following effects, for example:interfere with the expression of a cancer stem cell marker; interferewith activation of a cancer stem cell signal transduction pathway by,for example, sterically inhibiting interactions between a cancer stemcell marker and its ligand, receptor or co-receptors; or bind to acancer stem cell marker and trigger cell death or inhibit tumor cellproliferation.

In certain embodiments, the present invention provides antagonistsagainst a cancer stem cell marker that act extracellularly to affect orinhibit the function of a cancer stem cell marker. In certainembodiments, an antagonist is a small molecule that binds to theextracellular domain of a cancer stem cell marker protein. In otherembodiments, an antagonist of a cancer stem cell marker isproteinaceous. In some embodiments the proteinaceous antagonist is afragment or amino acid sequence variant of a native cancer stem cellmarker receptor or binding partner. In some embodiments the fragment oramino acid sequence variant can bind a cancer stem cell marker receptorto enhance or prevent binding of a signaling ligand. In otherembodiments the fragment or amino acid sequence variant of a nativecancer stem cell marker or binding partners can bind to the signalingligand of a cancer stem cell marker to enhance or prevent binding of thesignaling ligand. In some embodiments the antagonist is a soluble cancerstem cell protein receptor or soluble receptor protein. Extracellularbinding of an antagonist against a cancer stem cell marker can inhibitthe signaling of a cancer stem cell marker protein by inhibitingintrinsic activation (e.g. kinase activity) of a cancer stem cell markerand/or by sterically inhibiting the interaction, for example, of acancer stem cell marker with its ligand, of a cancer stem cell markerwith its receptor, of a cancer stem cell marker with a co-receptor, orof a cancer stem cell marker with the extracellular matrix. Furthermore,extracellular binding of an antagonist against a cancer stem cell markercan downregulate cell-surface expression of a cancer stem cell markersuch as, for example, by internalization of a cancer stem cell markerprotein and/or decreasing cell surface trafficking of a cancer stem cellmarker.

In certain embodiments, antagonists of a cancer stem cell marker cantrigger cell death indirectly by inhibiting angiogenesis. Angiogenesisis the process by which new blood vessels form from pre-existing vesselsand is a fundamental process required for normal growth, for example,during embryonic development, wound healing and in response toovulation. Solid tumor growth larger than 1-2 mm² also requiresangiogenesis to supply nutrients and oxygen without which tumor cellsdie. Thus in certain embodiments, an antagonist of a cancer stem cellmarker targets vascular cells that express the cancer stem cell markerincluding, for example, endothelial cells, smooth muscle cells orcomponents of the extracellular matrix required for vascular assembly.In some embodiments, an antagonist of a cancer stem cell marker inhibitsgrowth factor signaling required by vascular cell recruitment, assembly,maintenance or survival.

Polynucleotides

The invention is directed to isolated polynucleotides encoding thepolypeptides comprising SEQ ID NOS: 1-9. The polynucleotides of theinvention can be in the form of RNA or in the form of DNA with DNAincluding cDNA, genomic DNA, and synthetic DNA. The DNA can bedouble-stranded or single-stranded, and if single stranded can be thecoding strand or non-coding (anti-sense) strand. Thus, the term“polynucleotide encoding a polypeptide” encompasses a polynucleotidethat includes only coding sequences for the polypeptide as well as apolynucleotide which includes additional coding and/or non-codingsequences.

The present invention further relates to variants of the hereinabovedescribed polynucleotides that encode, for example, fragments, analogs,and derivatives. The variant of the polynucleotide can be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide. As hereinabove indicated, thepolynucleotide can have a coding sequence which is a naturally occurringallelic variant of the coding sequence of a disclosed polypeptide. Asknown in the art, an allelic variant is an alternate form of apolynucleotide sequence which has a substitution, deletion, or additionof one or more nucleotides, and which does not substantially alter thefunction of the encoded polypeptide.

The present invention also includes polynucleotides wherein the codingsequence for the mature polypeptide can be fused in the same readingframe to a polynucleotide which aids in, for example, expression,secretion, protein stability of a polypeptide from a host cellincluding, for example, a leader sequence which functions as a secretorysequence for controlling transport of a polypeptide from the cell. Thepolypeptide having a leader sequence is a preprotein and can have theleader sequence cleaved by the host cell to form the mature form of thepolypeptide. The polynucleotides can also encode for a proprotein whichis the mature protein plus additional 5′ amino acid residues. A matureprotein having a prosequence is a proprotein and is an inactive form ofthe protein. Once the prosequence is cleaved an active mature proteinremains. Thus, for example, the polynucleotide of the present inventioncan encode for a mature protein, or for a protein having a prosequenceor for a protein having both a prosequence and presequence (leadersequence).

The polynucleotides of the present invention can also have the codingsequence fused in-frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence can be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencecan be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell 37:767 (1984)).

Certain embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, 95% identical, and in some embodiments, at least96%, 97%, 98% or 99% identical to a nucleotide that encodes thedisclosed sequences.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

Soluble Receptor Polypeptides

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides having thesequence of SEQ ID NOS: 1-9 It will be recognized in the art that someamino acid sequences of the invention can be varied without significanteffect on the structure or function of the protein. If such differencesin sequence are contemplated, it should be remembered that there will becritical areas on the protein which determine activity. Thus, theinvention further includes variations of the polypeptides which showsubstantial activity or which include regions of FZD protein such as theprotein portions discussed herein. Such mutants include deletions,insertions, inversions, repeats, and type substitutions. As indicatedbelow, guidance concerning which amino acid changes are likely to bephenotypically silent can be found in Bowie, et al., Science247:1306-1310 (1990).

Thus, the fragments, derivatives, or analogs of the polypeptides of theinvention can be: (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue and such substituted amino acid residue can or can not be oneencoded by the genetic code; or (ii) one in which one or more of theamino acid residues includes a substituted group; or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol); or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives, andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the soluble receptor protein. Theprevention of aggregation is highly desirable, as aggregation orproteins not only results in a loss of activity but can also beproblematic when preparing pharmaceutical formulations, because they canbe immunogenic. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993)).

As indicated, changes are typically of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Tables 1 and 2).

TABLE 1 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

TABLE 2 Amino Acid Substitutions Original Residue SubstitutionsExemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R) Lys Lys; Gln;Asn Asn (N) Gln Gln; His; Lys; Arg Asp (D) Glu Glu Cys (C) Ser Ser Gln(Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Arg Asn; Gln; Lys;Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; norleucine Leu (L) Ilenorleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M)Leu Leu; Phe; Ile Phe (F) Leu Leu; Val; Ile; Ala Pro (P) Gly Gly Ser (S)Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; SerVal (V) Leu Ile; Leu; Met; Phe; Ala; norleucine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of substitutions for any given solublereceptor polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5or 3.

The polypeptides of the present invention include the polypeptides ofSEQ ID NOS: 1-9 as well as polypeptides which have at certain times atleast 90% similarity to the polypeptides of SEQ ID NOS: 1-9, and atcertain times at least 95% similarity to the polypeptides of SEQ ID NOS:1-9, and at certain times at least 96%, 97%, 98%, or 99% similarity tothe polypeptides of SEQ ID NOS: 1-9. As known in the art “similarity”between two polypeptides is determined by comparing the amino acidsequence and its conserved amino acid substitutes of one polypeptide tothe sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention canbe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments can be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention can be used tosynthesize full-length polynucleotides of the present invention.

A fragment of the proteins of this invention is a portion or all of aprotein which is capable of binding to a cancer stem cell marker proteinor cancer stem cell protein binding partner (e.g. a receptor,co-receptor, ligand, or co-ligand). This fragment has a high affinityfor a cancer stem cell marker protein or cancer stem cell proteinbinding partner (e.g. a receptor, co-receptor, ligand, or co-ligand).Some fragments of fusion proteins are protein fragments comprising atleast part of the extracellular portion of a cancer stem cell markerprotein or cancer stem cell protein binding partner bound to at leastpart of a constant region of an immunoglobulin. The affinity can be inthe range of about 10⁻¹¹ to 10⁻¹² M, although the affinity can varyconsiderably with fragments of different sizes, ranging from 10⁻⁷ to10⁻¹³ M. In some embodiments, the fragment is about 10-255 amino acidsin length and comprises the cancer stem cell marker protein ligandbinding site linked to at least part of a constant region of animmunoglobulin.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in Remington's PharmaceuticalSciences, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The chemical moieties most suitable for derivatization include watersoluble polymers. A water soluble polymer is desirable because theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. In some embodiments,the polymer will be pharmaceutically acceptable for the preparation of atherapeutic product or composition. One skilled in the art will be ableto select the desired polymer based on such considerations as whetherthe polymer/protein conjugate will be used therapeutically, and if so,the desired dosage, circulation time, resistance to proteolysis, andother considerations. The effectiveness of the derivatization can beascertained by administering the derivative, in the desired form (i.e.,by osmotic pump, or by injection or infusion, or, further formulated fororal, pulmonary or other delivery routes), and determining itseffectiveness. Suitable water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly 1,3 dioxolane, poly 1,3,6 trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde can have advantages in manufacturing due to itsstability in water.

The number of polymer molecules so attached can vary, and one skilled inthe art will be able to ascertain the effect on function. One canmono-derivatize, or can provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to protein (or peptide)molecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted protein or polymer) willbe determined by factors such as the desired degree of derivatization(e.g., mono-, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art. See for example, EP 0 401384, the disclosure of which is hereby incorporated by reference(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresylchloride). For example, polyethylene glycol can be covalently boundthrough amino acid residues via a reactive group, such as, a free aminoor carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule can be bound. The amino acid residueshaving a free amino group can include lysine residues and the N-terminalamino acid residue. Those having a free carboxyl group can includeaspartic acid residues, glutamic acid residues, and the C terminal aminoacid residue. Sulfhydryl groups can also be used as a reactive group forattaching the polyethylene glycol molecule(s). For therapeutic purposes,attachment at an amino group, such as attachment at the N-terminus orlysine group can be performed. Attachment at residues important forreceptor binding should be avoided if receptor binding is desired.

One can specifically desire an amino-terminal chemically modifiedprotein. Using polyethylene glycol as an illustration of the presentcompositions, one can select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)can be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminal chemicalmodification can be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivatization in aparticular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onecan selectively N-terminally pegylate the protein by performing thereaction at a pH which allows one to take advantage of the pKadifferences between the epsilon amino group of the lysine residues andthat of the alpha amino group of the N terminal residue of the protein.By such selective derivatization, attachment of a water soluble polymerto a protein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer can be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, can be used.

Pegylation can be carried out by any of the pegylation reactions knownin the art. See, for example: Focus on Growth Factors, 3(2): 4-10(1992); EP 0 154 316, the disclosure of which is hereby incorporated byreference; EP 0 401 384; and the other publications cited herein thatrelate to pegylation. The pegylation can be carried out via an acylationreaction or an alkylation reaction with a reactive polyethylene glycolmolecule (or an analogous reactive water soluble polymer).

Thus, it is contemplated that soluble receptor polypeptides to be usedin accordance with the present invention can include pegylated solublereceptor protein or variants, wherein the PEG group(s) is (are) attachedvia acyl or alkyl groups. Such products can be mono pegylated or polypegylated (e.g., containing 2 6, and typically 2 5, PEG groups). The PEGgroups are generally attached to the protein at the α or ε amino groupsof amino acids, but it is also contemplated that the PEG groups could beattached to any amino group attached to the protein, which issufficiently reactive to become attached to a PEG group under suitablereaction conditions.

The polymer molecules used in both the acylation and alkylationapproaches can be selected from among water soluble polymers asdescribed above. The polymer selected should be modified to have asingle reactive group, such as an active ester for acylation or analdehyde for alkylation, so that the degree of polymerization can becontrolled as provided for in the present methods. An exemplary reactivePEG aldehyde is polyethylene glycol propionaldehyde, which is waterstable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see, U.S.Pat. No. 5,252,714). The polymer can be branched or unbranched. For theacylation reactions, the polymer(s) selected should have a singlereactive ester group. For the present reductive alkylation, thepolymer(s) selected should have a single reactive aldehyde group.Generally, the water soluble polymer will not be selected from naturallyoccurring glycosyl residues since these are usually made moreconveniently by mammalian recombinant expression systems. The polymercan be of any molecular weight, and can be branched or unbranched. Onewater soluble polymer for use herein is polyethylene glycol. As usedherein, polyethylene glycol is meant to encompass any of the forms ofPEG that have been used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol.

Other reaction parameters, such as solvent, reaction times,temperatures, etc., and means of purification of products, can bedetermined case by case based on the published information relating toderivatization of proteins with water soluble polymers (see thepublications cited herein).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. For example, cDNA can be obtained by screening a humancDNA library with a labeled DNA fragment encoding the polypeptide of SEQID NO: 1 and identifying positive clones by autoradiography. Furtherrounds of plaque purification and hybridization are performed usingconventional methods.

In some embodiments of a recombinant method, a DNA sequence isconstructed by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional analogsthereof. See, e.g. Zoeller et al., Proc. Natl. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585. Another method ofconstructing a DNA sequence encoding a polypeptide of interest would beby chemical synthesis using an oligonucleotide synthesizer. Sucholigonucleotides can be designed based on the amino acid sequence of thedesired polypeptide and selecting those codons that are favored in thehost cell in which the recombinant polypeptide of interest will beproduced.

Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest. For example, acomplete amino acid sequence can be used to construct a back-translatedgene. Further, a DNA oligomer containing a nucleotide sequence codingfor the particular isolated polypeptide can be synthesized. For example,several small oligonucleotides coding for portions of the desiredpolypeptide can be synthesized and then ligated. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the mutant DNA sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

Recombinant expression vectors can be used to amplify and express DNAencoding cancer stem cell marker polypeptide fusions. Recombinantexpression vectors are replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a cancer stem cell markerpolypeptide fusion or a bioequivalent analog operatively linked tosuitable transcriptional or translational regulatory elements derivedfrom mammalian, microbial, viral or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a genetic element or elementshaving a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences, as described in detail below. Such regulatory elements caninclude an operator sequence to control transcription. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, operatively linkedmeans contiguous and, in the case of secretory leaders, means contiguousand in reading frame. Structural elements intended for use in yeastexpression systems can include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an N-terminal methionine residue. This residue canoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovims andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a cancer stem cell marker proteininclude prokaryotes, yeast, insect or higher eukaryotic cells under thecontrol of appropriate promoters. Prokaryotes include gram negative orgram positive organisms, for example E. coli or bacilli. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems could also be employed.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described by Pouwels etal., Cloning Vectors: A Laboratory Manual, Elsevier, N.Y. (1985), therelevant disclosure of which is hereby incorporated by reference.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman, Cell 23:175(1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a cancer stem cell protein-Fc composition.Some or all of the foregoing purification steps, in variouscombinations, can also be employed to provide a homogeneous recombinantprotein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Inhibiting Tumor Cell Growth

The present invention also provides methods for inhibiting the growth oftumorigenic cells expressing a cancer stem cell marker using theantagonists of a cancer stem cell marker described herein. In certainembodiments, the method of inhibiting the growth of tumorigenic cellsexpressing a cancer stem cell marker comprises contacting the cell withan antagonist against a cancer stem cell marker in vitro. For example,an immortalized cell line or a cancer cell line that expresses a cancerstem cell marker is cultured in medium to which is added an antagonistof the expressed cancer stem cell marker to inhibit cell growth.Alternatively tumor cells and/or tumor stem cells are isolated from apatient sample such as, for example, a tissue biopsy, pleural effusion,or blood sample and cultured in medium to which is added an antagonistof a cancer stem cell marker to inhibit cell growth. In certainembodiments, the antagonist is a cancer stem cell marker protein fusionthat specifically binds to a cancer stem cell marker protein or cancerstem cell marker binding protein (e.g. receptor, co-receptor, ligand, orco-ligand). For example, a purified cancer stem cell marker proteinfusion is added to the culture medium of isolated cancer stem cell toinhibit cell growth.

In certain embodiments, the method of inhibiting the growth oftumorigenic cells expressing a cancer stem cell marker comprisescontacting the cell with an antagonist against a cancer stem cell markerin vivo. In certain embodiments, contacting a tumorigenic cell with anantagonist to a cancer stem cell marker is undertaken in an animalmodel. For example, xenografts expressing a cancer stem cell marker aregrown in immunocompromised mice (e.g. NOD/SCID mice) that areadministered an antagonist to a cancer stem cell marker to inhibit tumorgrowth. Alternatively, cancer stem cells that express a cancer stem cellmarker are isolated from a patient sample such as, for example, a tissuebiopsy, pleural effusion, or blood sample and injected intoimmunocompromised mice that are then administered an antagonist againstthe cancer stem cell marker to inhibit tumor cell growth. In someembodiments, the antagonist of a cancer stem cell marker is administeredat the same time or shortly after introduction of tumorigenic cells intothe animal to prevent tumor growth. In some embodiments, the antagonistof a cancer stem cell marker is administered as a therapeutic after thetumorigenic cells have grown to a specified size. In some embodiments,the antagonist is a cancer stem cell marker protein fusion thatspecifically binds to a cancer stem cell marker protein or cancer stemcell marker binding protein (e.g. receptor, co-receptor, ligand, orco-ligand). In certain embodiments, contacting a tumorigenic cell withan antagonist to a cancer stem cell is undertaken in a human patientdiagnosed with cancer.

Pharmaceutical Compositions

The present invention further provides pharmaceutical compositionscomprising antagonists (e.g. antibodies) that target a cancer stem cellmarker. These pharmaceutical compositions find use in inhibiting tumorcell growth and treating cancer in human patients.

Formulations are prepared for storage and use by combining a purifiedantagonist (e.g. antibody) of the present invention with apharmaceutically acceptable carrier, excipient, and/or stabilizer as asterile lyophilized powder, aqueous solution, etc (Remington, TheScience and Practice of Pharmacy, 20th Edition, Mack Publishing (2000)).Suitable carriers, excipients, or stabilizers comprise nontoxic bufferssuch as phosphate, citrate, and other organic acids; salts such assodium chloride; antioxidants including ascorbic acid and methionine;preservatives (e.g. octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl orpropyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight polypeptides (such as less than about 10amino acid residues); proteins such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; carbohydrates such as monosaccharides, disaccharides,glucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN or polyethyleneglycol (PEG).

The pharmaceutical composition of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, or suppositoriesfor oral, parenteral, or rectal administration or for administration byinhalation. In solid compositions such as tablets the principal activeingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother diluents (e.g. water) to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention,or a non-toxic pharmaceutically acceptable salt thereof. The solidpreformulation composition is then subdivided into unit dosage forms ofthe type described above. The tablets, pills, etc of the novelcomposition can be coated or otherwise compounded to provide a dosageform affording the advantage of prolonged action. For example, thetablet or pill can comprise an inner composition covered by an outercomponent. Furthermore, the two components can be separated by anenteric layer that serves to resist disintegration and permits the innercomponent to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

Pharmaceutical formulations include antagonists of the present inventioncomplexed with liposomes (Epstein, et al., Proc. Natl. Acad. Sci. USA82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545). Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Liposomes can be generated by the reverse phase evaporation with a lipidcomposition comprising phosphatidylcholine, cholesterol, andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The antagonist can also be entrapped in microcapsules. Suchmicrocapsules are prepared, for example, by coacervation techniques orby interfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions as described in Remington, TheScience and Practice of Pharmacy, 20th Ed. Mack Publishing (2000).

In addition sustained-release preparations can be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles (e.g. films, ormicrocapsules). Examples of sustained-release matrices includepolyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

Treatment with Antagonists

It is envisioned that the antagonists of the present invention can beused to treat various conditions characterized by expression and/orincreased responsiveness of cells to a cancer stem cell marker.Particularly it is envisioned that the antagonists (e.g. antibodies)against a cancer stem cell marker will be used to treat proliferativedisorders including but not limited to benign and malignant tumors ofthe kidney, liver, bladder, breast, stomach, ovary, colon, rectum,prostate, lung, vulva, thyroid, head and neck, brain (glioblastoma,astrocytoma, medulloblastoma, etc), blood and lymph (leukemias andlymphomas).

The antagonists are administered as an appropriate pharmaceuticalcomposition to a human patient according with known methods. Suitablemethod of administration include intravenous administration as a bolusor by continuous infusion over a period of time include, but are notlimited to intramuscular, intraperitoneal, intravenous,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes.

In certain embodiments, the treatment involves the combinedadministration of an antagonist of the present invention and achemotherapeutic agent or cocktail of multiple differentchemotherapeutic agents. Treatment with an antagonist can occur priorto, concurrently with, or subsequent to administration ofchemotherapies. Chemotherapies contemplated by the invention includechemical substances or drugs which are known in the art and arecommercially available, such as Doxorubicin, 5-Fluorouracil, Cytosinearabinoside (“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine and Carboplatin.Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but preferably within a timeperiod such that all active agents can exert their biological activitiessimultaneously. Preparation and dosing schedules for suchchemotherapeutic agents can be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service, M. C. Perry, ed., Williams & Wilkins,Baltimore, Md. (1992).

In certain embodiments, the treatment involves the combinedadministration of an antagonist of the present invention and radiationtherapy. Treatment with an antagonist can occur prior to, concurrentlywith, or subsequent to administration of radiation therapy. Any dosingschedules for such radiation therapy can be used as determined by theskilled practitioner.

In certain embodiments, the treatment can involve the combinedadministration of antagonists of the present invention with antibodiesagainst additional tumor associated antigens including, but not limitedto, antibodies that bind to EGFR, HER2, and VEGF. Furthermore, treatmentcan include administration of one or more cytokines, can be accompaniedby surgical removal of cancer cells or any other therapy deemednecessary by a treating physician.

For the treatment of the disease, the appropriate dosage of anantagonist of the present invention depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the antagonist is administered for therapeutic orpreventative purposes, previous therapy, patient's clinical history, andso on all at the discretion of the treating physician. The antagonistcan be administered one time or over a series of treatments lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved (e.g. reduction in tumorsize). Optimal dosing schedules can be calculated from measurements ofdrug accumulation in the body of the patient and will vary depending onthe relative potency of an individual antagonist. The administeringphysician can easily determine optimum dosages, dosing methodologies andrepetition rates. In general, dosage is from 0.01 μg to 100 mg per kg ofbody weight, and can be given once or more daily, weekly, monthly oryearly. The treating physician can estimate repetition rates for dosingbased on measured residence times and concentrations of the drug inbodily fluids or tissues.

Kits

In yet other embodiments, the present invention provides kits that canbe used to perform the methods described herein. In certain embodiments,a kit comprises a purified cancer stem cell marker soluble receptor inone or more containers. In some embodiments, the kits contain all of thecomponents necessary and/or sufficient to perform a detection assay,including all controls, directions for performing assays, and anynecessary software for analysis and presentation of results. In certainembodiments, the present invention provides a compartment kit in whichreagents are contained in separate containers. Such containers allow oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the soluble receptor used in the methods,containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, etc.), and containers which contain the reagentsused to detect the hound antibody or probe. One skilled in the art willreadily recognize that the disclosed polynucleotides, polypeptides andantibodies of the present invention can be readily incorporated into oneof the established kit formats which are well known in the art.

EXAMPLES Example 1 Production of FZD Fc Soluble Receptor Proteins and InVivo Half-Life Determination

Soluble versions of the N-terminal extracellular domain (ECD) of humanFZD receptors bind Wnt ligands and act as antagonists of Wnt pathwaysignaling (He et al., (1997) Science 275:1652-54; Tanaka et al., (1998)Proc. Natl. Acad. Sci. 95:10164-69; Holmen et al., (2002) JBC277:34727-35; Vincan et al., (2005) Differentiation 73:142-53). SolubleFZD receptors were generated by ligating 1) the ECD or 2) the Fri domainof FZD10, FZD7, FZD5, FZD4, or FZD8 in-frame to human IgG₁ Fc isolatedfrom a human B-cell library (SEQ ID NO: 4) in a vector for expression ininsect cells and HEK 293 cells. Standard recombinant DNA technology wasused to isolate polynucleotides encoding FZD receptor ECDs including:amino acids from approximately 21 to 227 of FZD10 (FZD10 ECD.Fc); aminoacids from approximately 32 to 255 of FZD7 (FZD7 ECD.Fc); amino acidsfrom approximately 27 to 233 of FZD5 (FZD5 ECD.Fc); and amino acids fromapproximately 37 to 224 of FZD4 (FZD4 ECD.Fc) as well as FZD receptorFri domains including: amino acids from approximately 21 to 154 of FZD10(FZD10 Fri.Fc); amino acids from approximately 32 to 171 of FZD7 (FZD7Fri.Fc); amino acids from approximately 27 to 157 of FZD5 (FZD5 Fri.Fc);amino acids from approximately 37 to 170 of FZD4 (FZD4 Fri.Fc); andamino acids from approximately 28 to 158 of FZD8 (FZD8 Fri.Fc). Thesoluble receptor proteins were purified over a protein A column.

To determine the half-life of soluble FZD receptors, in vivo experimentswere performed. Specifically, 200 ug of purified FZD4 Fri.Fc, FZD8Fri.Fc, FZD5 Fri.Fc, and FZD5 ECD.Fc were administered i.p. to mice(n=3) and blood samples were obtained at indicated time points (FIG. 1).Serum proteins retained on Protein A agarose beads were separated on anSDS-PAGE gel, transferred to nitrocellulose membranes, and probed withHRP conjugated goat anti-human IgG Fc fragment to detect the hFc fusionproteins. FZD4 Fri.Fc, FZD5 Fri.Fc, and FZD8 Fri.Fc proteins are allpresent in blood serum 72 hours following injection, and FZD5 Fri.Fc andFZD8 Fri.Fc are present in blood serum 96 hours following injection(FIG. 1). In contrast, FZD5 ECD.Fc is undetectable after 24 hours (FIG.1).

Example 2 In Vitro Assays to Evaluate FZD Fc Soluble Receptor Protein

This example describes methods for in vitro assays to test the activityof FZD Fc receptor on cell proliferation and pathway activation.

Proliferation Assay

The expression of a FZD receptor by different cancer cell lines isquantified using Taqman analysis. Cell lines identified as expressing aFZD receptor are plated at a density of 10⁴ cell per well in 96-welltissue culture microplates and allowed to spread for 24 hours.Subsequently cells are cultured for an additional 12 hours in fresh DMEMwith 2% FCS at which point soluble FZD Fc receptor protein versuscontrol protein is added to the culture medium in the presence of 10umol/L BrdU. Following BrdU labeling, the culture media is removed, andthe cells fixed at room temperature for 30 min in ethanol and reactedfor 90 min with peroxidase-conjugated monoclonal anti-BrdU antibody(clone BMG 6H8, Fab fragments). The substrate is developed in a solutioncontaining tetramethylbenzidine and stopped after 15 min with 25 ul of 1mol/L H₂SO₄. The color reaction is measured with an automatic ELISAplate reader using a 450 nm filter (UV Microplate Reader; Bio-RadLaboratories, Richmond, Calif.). All experiments are performed intriplicate. The ability of FZD Fc soluble receptor protein to inhibitcell proliferation compared is determined.

Pathway Activation Assay

The ability of soluble FZD Fc receptor protein to block activation ofthe Wnt signaling pathway is determined in vitro. In one embodiment, HEK293 cells cultured in DMEM supplemented with antibiotics and 10% FCS areco-transfected with 1) Wnt713 and FZD10 expression vectors to activatethe Wnt signaling pathway; 2) a TCF/Luc wild-type or mutant reportervector containing three copies of the TCF-binding domain upstream of afirefly luciferase reporter gene to measure canonical Wnt signalinglevels (Gazit et al., 1999, Oncogene 18:5959-66); and 3) a Renillaluciferase reporter (Promega; Madison, Wis.) as an internal control fortransfection efficiency. FZD Fc protein is then added to the cellculture medium. Forty-eight hours following transfection, luciferaselevels are measured using a dual luciferase assay kit (Promega; Madison,Wis.) with firefly luciferase activity normalized to Renilla luciferaseactivity. Three independent experiments are preformed in triplicate. Theability of soluble FZD10 Fc protein to inhibit Wnt pathway activation isthus determined.

In some embodiments, increasing amounts of FZD Fc fusion proteins wereincubated with L cells in the presence or absence of Wnt3a ligand andthe Wnt3a induced stabilization of β-catenin was determined byimmunoblotting. Only in the presence of Wnt3a was β-catenin detectable,and this stabilization was blocked by increasing amounts of FZD5 ECD.Fc,FZD8 Fri.Fc and FZD4 Fri.Fc soluble receptor protein (FIG. 2)demonstrating that FZD Fc soluble receptor proteins antagonize Wntpathway signaling activated by the Wnt3a ligand.

The ability of FZD:Fc fusion proteins to antagonize signaling bydifferent Wnt ligands was then determined. HEK 293 cells stablytransfected with 8×TCF-luciferase reporter were incubated withincreasing amounts of FZD Fri.Fc soluble receptors in the presence ofdifferent Wnt ligands including Wnt1, Wnt2, Wnt3, Wnt3a and Wnt7b. FZD4Fri.Fc, FZD5 Fri.Fc and FZD8 Fri.Fc fusion proteins inhibited Wntsignaling mediated by all five Wnt ligands (FIG. 3).

Example 3 In Vivo Prevention of Tumor Growth Using FZD Fc SolubleReceptor Protein

This example describes the use of a FZD Fc soluble receptor to preventtumor growth in a xenograft model.

Tumor cells from a patient sample (solid tumor biopsy or pleuraleffusion) that have been passaged as a xenograft in mice were preparedfor repassaging into experimental animals as described in detail above.Dissociated tumor cells (<10,000 cells per animal; n=10) were theninjected subcutaneously into the mammary fat pads NOD/SCID mice toelicit tumor growth.

In certain embodiments, dissociated tumor cells are first sorted intotumorigenic and non-tumorigenic cells based on cell surface markersbefore injection into experimental animals. Specifically, tumor cellsdissociated as described above are washed twice with Hepes bufferedsaline solution (HBSS) containing 2% heat-inactivated calf serum (HICS)and resuspended at 10⁶ cells per 100 ul. Antibodies are added and thecells incubated for 20 min on ice followed by two washes with HBSS/2%HICS. Antibodies include anti-ESA (Biomeda, Foster City, Calif.),anti-CD44, anti-CD24, and Lineage markers anti-CD2, -CD3, -CD10, -CD16,-CD18, -CD31, -CD64, and -CD140b (collectively referred to as Lin;PharMingen, San Jose, Calif.). Antibodies are directly conjugated tofluorochromes to positively or negatively select cells expressing thesemarkers. Mouse cells are eliminated by selecting against H2Kd+ cells,and dead cells are eliminated by using the viability dye 7AAD. Flowcytometry is performed on a FACSVantage (Becton Dickinson, FranklinLakes, N.J.). Side scatter and forward scatter profiles are used toeliminate cell clumps. Isolated ESA+, CD44+, CD24−/low, Lin− tumorigeniccells are then injected subcutaneously into the mammary fat pads forbreast tumors or into the flank for non-breast tumors of NOD/SCID miceto elicit tumor growth.

In certain embodiments, two days after tumor cell injection, the animalswere treated with FZD7 ECD.Fc soluble receptor, FZD10 ECD.Fc solublereceptor, or FZD5 ECD.Fc soluble receptor. Each test injected animalreceived 10 mg/kg FZD7 ECD.Fc, FZD5 ECD.Fc or FZD10 ECD.Fc proteinintraperitoneal (i.p.) 2-3× per week for a total of 4 weeks. Controlinjected animals were injected 2× per week for a total of 4 weeks. Tumorsize was assessed on days 21, 24, 28, and 30. Treatment with bothsoluble FZD10 ECD.Fc and FZD7 ECD.Fc reduced total tumor volume comparedto control treated animals (FIG. 4). The reduction of tumor volume byFZD7 ECD.Fc was statistically significant on day 28 and day 30 (FIG. 4).

Next the effect of FZD Fc soluble receptor treatment on the presence ofcancer stem cells in a tumor is assessed. Tumor samples from FZD Fcversus control treated mice are cut up into small pieces, mincedcompletely using sterile blades, and single cell suspensions obtained byenzymatic digestion and mechanical disruption. Dissociated tumor cellsare then analyzed by FACS analysis for the presence of tumorigeniccancer stem cells based on ESA+, CD44+, CD24−/low, Lin− surface cellmarker expression as described in detail above.

The tumorigenicity of cells isolated based on ESA+, CD44+, CD24−/low,Lin− expression following FZD Fc treatment can then assessed. 5,000,1,000, 500, and 100 isolated ESA+, CD44+, CD24−/low, Lin− cancer stemcells from FZD Fc treated versus control treated mice are re-injectedsubcutaneously into the mammary fat pads of NOD/SCID mice. Thetumorigenicity of cancer stem cells based on the number of injectedcells required for consistent tumor formation is thus determined.

In certain embodiments, female rag-2/γ chain double knockout mice wereinjected at age 5-7 weeks with 50,000 mouse mammary tumor virus(MMTV)-WNT1 tumor derived cells in the upper right mammary fat pad.Transgenic (MMTV)-Wnt-1 mice exhibit discrete steps of mammarytumorigenesis, including hyperplasia, invasive ductal carcinoma, anddistant metastasis, and thus this mouse model of breast cancer providesa useful tool for analyzing the role of Wnts in tumor formation andgrowth (Nusse and Varmus (1982) Cell 31:99-109). Tumors from these micewere dissociated and these dissociated tumor cells used for tumorpropagation purposes. Mice with tumor cells implanted in the mammary fatpad were treated 5× weekly with 200 ul PBS (n=10) or FZD8 Fri.Fc solublereceptor (10 mg/kg) diluted in PBS. Once tumors were palpable, tumorsizes were measured twice weekly. Treatment with soluble receptor FZD8Fri.Fc dramatically reduced the growth of tumors compared to the controltreatment with PBS (FIG. 5).

To again test the ability of FZD soluble receptors to inhibit tumorgrowth, NOD/SCID mice were injected with 50,000 PE13 breast tumor cells.One day following cell injection, 200 ul FZD8 Fri.Fc soluble receptordiluted in PBS was injected i.p. at 10 mg/kg or 200 ul PBS was injectedand treatment was continued 5× weekly (n=10 per experimental group).Tumor growth was monitored weekly until growth was detected, then tumorgrowth was measured twice weekly. Treatment of animals with FZD8 Fri.Fcsignificantly reduced breast tumor cell growth compared to PBS injectedcontrols (FIG. 6).

Example 4 In Vivo Treatment of Tumor Growth Using FZD Fc SolubleReceptor Protein

This example describes the use of a FZD Fc soluble receptor to treattumors in a xenograft model.

In certain embodiments, 50,000 MMTV Wnt1 breast tumor derived cells inMatrigel were sub-cutaneously implanted into 5-7 week old female rag-2/γchain double knockout mice. On day nineteen, mice with tumors wererandomly assigned to groups with a mean tumor volume of 65 mm³, and onday twenty-six, treatment with FZD8 Fri.Fc or FZD5 Fri.Fc fusionproteins was initiated. Specifically, five times per week FZD8 Fri.Fcfusion protein was administered at increasing concentrations (5 mg/kg,10 mg/kg, and 30 mg/kg), and FZD5 Fri.Fc was administered at 10 mg/kg.Control animals were treated with PBS.

A dose dependent anti-tumor activity of FZD8 Fri.Fc fusion protein wasobserved (FIG. 7). At the lowest dose—5 mg/kg—FZD8 Fri.Fc reduced thegrowth of tumors relative to mice treated with PBS, but the 10 mg/kg and30 mg/kg FZD8 Fri.Fc treatment regimens were significantly moreeffective in reducing the size of the pre-established tumors. Incontrast, FZD5 Fri.Fc did not display anti-tumor effects on establishedbreast tumors that require wnt1 for growth.

Example 5 In Vivo Treatment of Tumors Using FZD Fc Soluble ReceptorProtein

This example describes the use of a FZD Fc soluble receptor to treatcancer in a xenograft model.

Tumor cells from a patient sample (solid tumor biopsy or pleuraleffusion) that have been passaged as a xenograft in mice are preparedfor repassaging into experimental animals. Tumor tissue is removed, cutup into small pieces, minced completely using sterile blades, and singlecell suspensions obtained by enzymatic digestion and mechanicaldisruption. Dissociated tumor cells are then injected subcutaneouslyinto the mammary fat pads for breast tumors or into the flank fornon-breast tumors NOD/SCID mice to elicit tumor growth. Alternatively,ESA+, CD44+, CD24−/low, Lin− tumorigenic tumor cells are isolated asdescribed in detail above and injected.

Following tumor cell injection, animals are monitored for tumor growth.Once tumors reach an average size of approximately 150 to 200 mm, FZD Fcprotein treatment begins. Each animal receives 10 mg/kg FZD Fc orcontrol protein i.p. two to five times per week for a total of 6 weeks.Tumor size is assessed twice a week during these 6 weeks. The ability ofFZD Fc to prevent further tumor growth or to reduce tumor size comparedto control antibodies is thus determined.

Example 6 Treatment of Human Cancer Using FZD Fc Soluble ReceptorProtein

This example describes methods for treating cancer using a FZD Fcsoluble receptor to target tumors comprising cancer stem cells and/ortumor cells in which FZD receptor expression has been detected.

The presence of cancer stem cell marker expression can first bedetermined from a tumor biopsy. Tumor cells from a biopsy from a patientdiagnosed with cancer are removed under sterile conditions. In oneembodiment the tissue biopsy is fresh-frozen in liquid nitrogen,embedded in O.C.T., and cut on a cryostat as 10 um sections onto glassslides. Alternatively the tissue biopsy is formalin-fixed,paraffin-embedded, and cut on a microtome as 10 um section onto glassslides. Sections are incubated with antibodies against a FZD receptor todetect protein expression. Additionally, the presence of cancer stemcells can be determined. Tissue biopsy samples are cut up into smallpieces, minced completely using sterile blades, and cells subject toenzymatic digestion and mechanical disruption to obtain a single cellsuspension. Dissociated tumor cells are then incubated with anti-ESA,-CD44, -CD24, -Lin, and -FZD antibodies to detect cancer stem cells, andthe presence of ESA+, CD44+, CD24−/low, Lin−, FZD+ tumor stem cells isdetermined by flow cytometry as described in detail above.

Cancer patients whose tumors are diagnosed with cancer stem cells aretreated with a FZD:Fc soluble receptor. Human FZD Fc fusion proteingenerated as described above is purified and formulated with a suitablepharmaceutical carrier in PBS for injection. Patients are treated withFZD Fc preferably once a week for at least 10 weeks, but more preferablyonce a week for at least about 14 weeks. Each administration of FZD Fcshould be a pharmaceutically effective dose of about 2 to about 100mg/ml or about 5 to about 40 mg/ml. FZD Fc can be administered prior to,concurrently with, or after standard radiotherapy regimens orchemotherapy regimens using one or more chemotherapeutic agent, such asoxaliplatin, fluorouracil, leucovorin, or streptozocin. Patients aremonitored to determine whether such treatment has resulted in ananti-tumor response, for example, based on tumor regression, reductionin the incidences of new tumors, lower tumor antigen expression,decreased numbers of cancer stem cells, or other means of evaluatingdisease prognosis.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in the relevant fieldsare intended to be within the scope of the following claims.

FZD10 N-terminal extracellular domain SEQ ID NO: 1MQRPGPRLWLVLQVMGSCAAISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIIVIEQFNEKWPDSLDCRKLPNKNDPNYLCMEAPNNGSDEPTRGSGLEPPLFRPQRPHSAQEHPLKDGGPGRGGCDNPGKFHHVEKSASCAPLCTPGVDVYWSREDKRFA FZD7 N-terminal extracellular domainSEQ ID NO: 2 MRDPGAAAPLSSLGLCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLPDLPFTALPPGASDGRGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRANGLMYFKEEER RFARLFZD5 N-terminal extracellular domain SEQ ID NO: 3MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTIIMPNQFNIEDIQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPIMRQYGMWPERMSCDRLPVLGRDAEVLCMDYNRSEAFIAPPRPFPAKPTLPGPPGAPASGGECPAGGPFVCKCREPFVPILKESHPLYNKVRTGQVPNCAVPCYQPSFSADERT Human IgG₁ Fc SEQ ID NO: 4DKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FZD6 N-terminal extracellular domainSEQ ID NO: 5 MEMFTELLTCIFLPLLRGHSLETCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVFTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGPQKKTEQVQRDIGEWCPRHLKTSGGQGYKFLGIDQCAPPCPNMYEKSDELEFAKS FIGTVSIFZD N-terminal extracellular domain SEQ ID NO: 6MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGEGDEEERRCDPIRISMCQNLGYNVIKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCISVKREWILPVLKEFGFAWPESLNCSKEPPQNDHNHMCMEGPGDEEVPLPHKTPIQPCIEECIISVGTNSDQYIWVKRSLNCVLKCGYDAGLYSRSAKEFTDI FZD8 Fri domain SEQ ID NO: 7MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMD YNRTDLTTFZD4 Fri domain SEQ ID NO: 8MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSK FPPQNDHNHMCMEGPGDEEVFZD5 Fri domain SEQ ID NO: 9MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHOTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDY NRSEATT

What is claimed is:
 1. A method of treating cancer in a human subject inneed thereof, the method comprising administering to the subject asoluble receptor comprising (a) a fragment of an extracellular domain ofa human Frizzled (FZD) receptor and (b) a human Fc domain, wherein: (i)the fragment of the extracellular domain of the human FZD receptorconsists essentially of the Fri domain of the human FZD receptor; (ii)the soluble receptor has a longer half-life in vivo than a solublereceptor comprising the extracellular domain of the FZD receptor and thehuman Fc, domain; and (iii) the cancer is selected from the groupconsisting of pancreatic cancer, ovarian cancer, hepatocellular cancer,breast cancer, colorectal cancer, lung cancer, prostate cancer, and headand neck cancer.
 2. The method of claim 1, wherein the cancer isselected from the group consisting of ovarian cancer, pancreatic cancer,and hepatocellular cancer.
 3. The method of claim 1, wherein the Fridomain of the human FZD receptor comprises the Fri domain of FZD4, FZD5,or FZD8.
 4. The method of claim 3, wherein the Fri domain of the humanFZD receptor comprises the Fri domain of FZD8.
 5. The method of claim 4,wherein the Fri domain of human FZD8 comprises the amino acids fromapproximately 28 to 158 of the amino acid sequence of SEQ NO:7.
 6. Themethod of claim 5, wherein the human Fe domain is human IgG1 Fccomprising the amino acid sequence of SEQ ID NO:4.
 7. The method ofclaim 6, wherein the cancer is ovarian cancer.
 8. The method of claim 7,wherein the soluble receptor is administered with chemotherapy.
 9. Themethod of claim 8, wherein the chemotherapy includes one or more ofpaclitaxel, cisplatin, and carboplatin.
 10. The method of claim 9,wherein the chemotherapy includes paclitaxel and carboplatin.
 11. Themethod of claim 6, wherein the cancer is pancreatic cancer.
 12. Themethod of claim 11, wherein the soluble receptor is administered withchemotherapy.
 13. The method of claim 6, wherein the cancer ishepatocellular cancer.
 14. The method of claim 13, wherein the solublereceptor is administered with chemotherapy.
 15. A method of treatingcancer in a human subject in need thereof, the method comprisingadministering to the subject a soluble receptor comprising a polypeptidethat consists of (a) a fragment of an extracellular domain of a humanFrizzled (FZD) receptor and (b) a human Fc domain, wherein: (i) thefragment of the extracellular domain of the human FZD receptor consistsessentially of the Fri domain of the human FZD receptor; (ii) the canceris selected from the group consisting of pancreatic cancer, ovariancancer, hepatocellular cancer, breast cancer, colorectal cancer, lungcancer, prostate cancer, and head and neck cancer.
 16. The method ofclaim 15, wherein the cancer is selected from the group consisting ofovarian cancer, pancreatic cancer, and hepatocellular cancer.
 17. Themethod of claim 15, wherein the Fri domain of the human FZD receptorcomprises the Fri domain of FZD4, FZD5, or FZD8.
 18. The method of claim17, wherein the Fri domain of the human FZD receptor comprises the Fridomain of FZD8.
 19. The method of claim 18, wherein the Fri domain ofhuman FZD8 comprises the amino acids from approximately 28 to 158 of theamino acid sequence of SEQ ID NO:7.
 20. The method of claim 19, whereinthe human Fc domain is human IgG1 Fc comprising the amino acid sequenceof SEQ ID NO:4.
 21. The method of claim 20, wherein the cancer isovarian cancer.
 22. The method of claim 21, wherein the soluble receptoris administered with chemotherapy.
 23. The method of claim 22, whereinthe chemotherapy includes one or more of paclitaxel, cisplatin, andcarboplatin.
 24. The method of claim 23, wherein the chemotherapyincludes paclitaxel and carboplatin.
 25. The method of claim 20, whereinthe cancer is pancreatic cancer.
 26. The method of claim 25, wherein thesoluble receptor is administered with chemotherapy.
 27. The method ofclaim 20, wherein the cancer is hepatocellular cancer.
 28. The method ofclaim 27, wherein the soluble receptor is administered withchemotherapy.